Composition and biosynthesis of sterols in selected marine phytoplankton

Six species of phytoplankton,Pseudoisochrysis paradoxa, Isochrysis galbana, Monochrysis lutheri, Platymonas suecica, Thalassiosira fluviatilis and aChaetoceros species, were cultured in the laboratory and their sterol contents analyzed utilizing digitonin precipitation, thin layer and gas chromatography and gas chromatography-mass spectrometry. A total of 7 sterols were found in phytoplankton. The occurrence of these sterols, cholest-5-en-3β-ol, cholest-5,22-dien-3β-ol, 24-methylcholesta-5,24(28)-dien-3β-ol, 24-methylcholest-5-en-3β-ol, 24-methylcholesta-5,22-dien-3β-ol, 24-ethylcholest-5-en-3β-ol and 24-ethylcholest-5,22-dien-3β-ol, differed significantly among the various phytoplankton species. Cultures ofP. paradoxa biosynthesized both of the sterols found in this species when incubated in the presence of¹⁴C- or³H-mevalonic acid for 0.5–9 days. These sterols were cholesterol and 24-methylcholesta-5,22-dien-3β-ol. Since 5 of the sterols found in the phytoplankton commonly occur in mollusks which feed on phytoplankton, it is likely that at least some of the tissue sterols in mollusks are of dietary origin. Research trainee, HL 07295-02, National Heart, Lung and Blood Insitute.     

Cooccurrence of C-24 alkylated Δ7- and Δ5-Sterols in the leaves ofBeta vulgarisin the leaves ofBeta vulgaris

The 4-desmethylsterols from the leaves ofBeta vulgaris are a mixture of Δ⁷-sterols (71%) and Δ⁵-sterols (29%). The Δ⁷-sterols isolated are spinasterol (24α-ethylcholesta-7,22-dien-3β-ol; 45%), 22-dihydrospinasterol (24α-ethylcholest-7-en-3β-ol; 24%), and avenasterol (24-ethylcholesta-7,24(28)-dien-3β-ol; 1.5%). The Δ⁵-sterols isolated are sitosterol (24α-ethylcholest-5-en-3β-ol; 15%), 24ζ-ethylcholesta-5,22-dien-3β-ol (7.5%), and 24ζ-methylcholest-5-en-3β-ol (7%).     

Sterols of scallop. III. Characterization of some C-24 epimeric sterols by high resolution (220 MHz) nuclear magnetic resonance spectroscopy

Alkyl sterols epimeric at C-24 isolated from the Atlantic scallop,Placopecten magellanicus, were analyzed by high resolution (220 MHz) nuclear magnetic resonance spectrometry, and their spectra compared with authentic samples. This technique was used to assign absolute stereochemistry in epimeric mixtures of 24 R and S 24-methylcholest-5-en-3β-ol, (22E)-24-methylcholesta-5,22-dien-3β-ol and 24-ethylcholest-5-en-3β-ol. It also allowed a semiquantitative estimate of the R/S isomers present in the mixture. M.S.R.L. Contribution Number 242.     

Sterols of the oyster,Crassostrea virginica

A commericial sample of the oyster,Crassostrea virginica, obtained from Maryland waters of the Chesapeake Bay, contained 31 desmethylesterols and at least eight 4-monomethylsterols. The combined gas liquid chromatography-mass spectra of the minor components showed the presence of 6 unusual sterols, 24-ethylcholest-22-en-3β ol, 4α-methyl-24-ethylcholestan-3β-ol, occelasterol, (24E)-24-propylidene-cholest-5-en-3β-ol, (24ZO-24-propylidene-cholest-5-en-3β-ol, and 24-methylene-cholestanol. The C-24 configuration of 24-ethylcholest-5-enol, 24-methyl-cholesta-5,22-dienol, and 24-ethylcholesta-5,22 dienol were elucidated by 220 MHz nuclear magnetic resonance spectrometry.     

Identification of 27-nor-(24R)-24-methylcholesta-5,22-dien-3β-ol and brassicasterol as the major sterols of the marine dinoflagellateGymnodinium simplex

The major 4α-monomethyl sterol of the dinoflagellateGymnodinium simplex was identified as (24S)-4α,24- dimethylcholestan-3β-ol. The major 4-demethyl sterols were characterized as (24R)-24-methylcholesta-5,22-dien-3β-ol (brassicasterol) and 27-nor-(24R)-24-methylcholesta-5,22-dien-3β-ol. The latter sterol has the opposite configuration at C-24 to that assigned to occelasterol, which has the same basic structure and has previously been reported as a constituent of the sterols of a marine worm. 24-Nor-cholesta-5,22-dien-3β-ol was also identified along with several other trace sterols. The co-occurrence of 27-nor-(24R)-cholesta-5,22-dien-3β-ol together with 24-nor-cholesta-5,22-dien-3β-ol and brassicasterol provides new evidence for the biosynthetic origins of the two former nor-sterols. It is suggested that they may be produced de novo by a route involving nor-isoprenoid pyrophosphates and nor-squalene as intermediates, rather than as bacterial degradation products of brassicasterol (or related sterols) as previously suggested in the literature. Presented at the “Sterol Symposium” of the American Oil Chemists' Society Annual International Congress, New Orleans, LA, May 1981.     

Identification of the free and conjugated sterol in a non-photosynthetic diatom,Nitzschia alba,as 24-methylene cholesterol

Previous studies on the sterol fraction of the nonphotosynthetic marine diatom,Nitszchia alba, indicated the major sterol to be either brassicasterol (24R-methylcholesta-5,22-dien-3β-ol) or 22-dehydrocampesterol (24S-methylcholesta-5,22-dien-3β-ol) on the basis only of gas chromatographymass spectral analysis. The present studies using nuclear magnetic resonance, infrared, and gas chromatography-mass spectrometry on the free and bound sterol fractions isolated by preparative thin layer chromatography showed the presence in both fractions of a single sterol, with spectral and chromatographic properties identical with those reported for 24-methylenecholesterol (ergosta-5,24(28)-dien-3β-ol). This sterol may be the precursor of 24-methyl sterols found in diatoms. The bound sterol fraction was found to consist of a single compound identified as 24-methylenecholesterol sulfate. No sterol esters or sterol glycosides were detected. Presented at symposium on Marine Lipids, AOCS Meeting, New York, May 1977.     

Diversity of sterol composition in the family chenopodiaceae

The predominant 4-desmethylsterols from the leaves of 13 species in eight genera of the family Chenopodiaceae are 24α-ethylsterols. In four species,Chenopodium ambrosioides L.,C. rubrum L.,Salicornia europaea L. andS. bigelovii Torr., the C-22(23) double bond is introduced into more than 70% of the 24α-ethylsterols producing spinasterol (24α-ethylcholesta-7,22E-dien-3β-ol) in the first two species and mixtures of spinasterol and stigmasterol (24α-ethylcholesta-5,22E-dien-3β-ol) in the latter species. The saturated side chain analogues predominate with more than 70% of the 24α-ethylsterols in eight species.Salsola kali L.,Suaeda linearis (Ell.) Moq.,Kochia scoparia (L.) Roth., andBassia hirsute (L.) Aschers. synthesize sitosterol (24α-ethylcholest-5-en-3β-ol), andAtriplex arenaria Nutt.,C. album L.,C. urbicum L. andC. leptophyllum Nutt. possess mixtures of sitosterol and 22-dihydrospinasterol (24α-ethylcholest-7-en-3β-ol). Sitostanol (24α-ethyl-5α-cholestan-3β-ol) was isolated fromSuaeda linearis as an 18% component of the total 4-desmethylsterol and in lesser amounts from four other species. In all species synthesizing 24-ethyl-Δ⁵-sterols, a 24ξ-methylcholest-5-en-3β-ol was also present at 1.0–20% of the total 4-desmethylsterol. Avenasterol [24-ethylcholesta-7,24(28)Z-dien-3β-ol], isofucosterol [24-ethylcholesta-5,24(28)Z-dien-3β-ol), cholesterol (cholest-5-en-3β-ol) and 24ξ-methyl-5α-cholestan-3β-ol also were isolated from several species. Species in the family Chenopodiaceae and the type genusChenopodium may be categorized into one of three groups based on sterol biosynthesis: the Δ⁷-sterol producers; the Δ⁵-sterol producers, and those producing mixtures of both Δ⁷- and Δ⁵-sterols in relatively fixed percentage compositions.     

Dominance of Δ7-sterols in the family caryophyllaceaein the family caryophyllaceae

The predominant 4-desmethylsterols from the leaves of 12 species in 11 genera of the family Caryophyllaceae are 24-ethyl-Δ⁷-sterols. In eight species,Scleranthus annus L.,Paronychia virginica Spreng.,Lychnis alba Mill.,Silene cucubalus Wibel,Dianthus armeria L.,Gypsophilia paniculata L.,Saponaria officinales L. andMyosoton aquaticum (L.) Moench, the major sterols are spinasterol (24α-ethylcholesta-7,22E-dien-3β-ol) and 22-dihydrospinasterol (24α-ethylcholest-7-en-3β-ol), with spinasterol at more than 60% of the desmethylsterol in the latter six species. Both 24α-and 24β-ethyl-Δ⁷-sterols are present in two species,Minuartia caroliniana Walt. andSpergula arvensis L., which possess 24β-ethylcholesta-7,25(27)-dien-3β-ol and 24β-ethylcholesta-7,22E,25(27)-trien-3β-ol as well as spinasterol and 22-dihydrospinasterol.Cerastium arvense L.,C. vulgatum L. andArenaria serpyllifolia L. possess 24-alkyl-Δ⁵ and Δ⁷-sterols. These three species synthesize sitosterol (24α-ethylcholest-5-en-3β-ol), 24ζ-methylcholest-5-en-3β-ol, spinasterol, 22-dihydrospinasterol and the stanols, sitostanol (24α-ethyl-5α-cholestan-3β-ol) and 24ζ-methyl-5α-cholestan-3β-ol. Avenasterol (24-ethylcholesta-7,24(28)Z-dien-3β-ol) was also isolated from five species. Sterol biosynthetic capability may be a useful characteristic in examining the taxonomic relatedness of plants in the Caryophyllaceae.     

Differences in the sterol composition of dominant antarctic zooplankton

The composition of free sterols was determined in Antarctic zooplankton species with various feeding behaviors. In the Southern Ocean, the dominant calanoid copepods Calanoides acutus, Calanus propinquus, Metridia gerlachei, and Euchaeta antarctica were investigated during different seasons and compared with the euphausiids Euphausia superba, E. crystallorophias, and Thysanoessa macrura. In addition, the Arctic copepods Calanus hyperboreus, C. glacialis, and C. finmarchicus were studied for comparison. Analyses were performed using gas chromatography and mass spectrometry. The zooplankton species exhibited a simple sterol content of up to six sterols. In the copepods, cholest-5-en-3β-ol (22.1 to 60.5%, range of sample means), cholesta-5,24-dien-3β-ol (22.3 to 45.2%), and cholesta-5,22E-dien-3β-ol (4.3 to 33.4%) contributed most, while in euphausiids the sterol composition was less complex with cholest-5-en-3β-ol always accounting for more than 75% of the total. Although sterols are membrane constituents and are expected not to vary considerably, differences in the abundance of sterols were observed between the species and the seasons. In herbivorous copepods, cholesta-5,24-dien-3β-ol increased by a factor of 1.5 to about 45% during the main feeding period in summer; this sterol is a metabolic precursor of cholest-5-en-3β-ol in the process of the dealkylation of dietary C-24 alkylated phytosterols. Cholest-5-en-3β-ol decreased by the same proportion. Omnivorous and carnivorous copepods showed average levels of cholesta-5,24-dien-3β-ol below 25%. These changes in sterol composition between copepod species seem to reflect their different feeding modes.     

Metabolism of sitosteryl β-D-glucoside and its nutritional effects in rats

[4-¹⁴C]Sitosteryl β-D-glucoside, intragastrically administered to rats, was not absorbed by the intestinal mucosa. At three hr after the application, radioactivity was concentrated almost exclusively in the digesta of stomach, small intestine as well as cecum and colon, whereas only low proportions of radioactively labeled compounds were found in the various tissues of the gastrointestinal tract. Minor proportions of labeled metabolites of [4-¹⁴C]sitosteryl β-D-glucoside, such as sitosterol and sitosteryl esters, were formed in the small intestine in vivo and in slices of small intestine in vitro. In the tissues of cecum and colon as well as the digesta derived from them, high proportions of labeled coprositostanol, i.e. 24α-ethyl-5β-cholestan-3β-ol, that obviously had been formed by bacterial degradation of the substrate were detected. The feeding of sitosteryl β-D-glucoside (0.5 g/kg body weight×day) over a period of four weeks did not alter significantly body weights or organ weights of rats. Analyses of steryl lipids of the various organs and tissues confirmed the findings obtained with the radioactive substrate: neither sitosteryl β-D-glucoside nor sitosterol or sitosteryl esters derived therefrom had been transported in appreciable amounts to organs and tissues outside the alimentary canal during the feeding period. Minor proportions of unmetabolized sitosteryl β-D-glucoside were detected in the tissues of stomach and intestine, whereas large proportions of the substrate were found in feces of rats that had received the sitosteryl β-D-glucoside-containing diet; coprositostanol was found in feces of these animals in high proportions as well. Thus, the use of sitosteryl β-D-glucoside as emulsifier or preservative in food and feed does not appear to involve any risk. The systematic nomenclature of the sterols referred to by trivial names is, cholest-5-en-3β-ol (cholesterol); 5α-cholestan-3β-ol (5α-cholestanol); 5β-cholestan-3β-ol (5β-cholestanol, coprostanol); 24α-methylcholest-5-en-3β-ol (campesterol); 24α-methyl-5α-cholestan-3β-ol (5α-campestanol); 24α-methyl-5β-cholestan-3β-ol (5β-campestanol, coprocampestanol); 24α-methyl-cholesta-5,22-dien-3β-ol (brassicasterol); 24α-ethylcholest-5-en-3β-ol (sitosterol, β-sitosterol); 24α-ethyl-5α-cholestan-3β-ol (5α-sitostanol); 24α-ethyl-5β-cholestan-3β-cholestan-3β-ol (5β-sitostanol, coprositostanol); 24α-ethylcholesta-5,22-dien-3β-ol (stigmasterol).     

New phospholipid fatty acids from the Caribbean spongeEctyoplasia ferox

The phospholipid fatty acids from the Caribbean spongeEctyoplasia ferox were studied. The novel fatty acids 25-methyl-5,9-heptacosadienoic (1) and 26-methyl-5,9-heptacosadienoic (2) were identified in 3.4 and 2.0% abundance, respectively, representing the longest set of Δ5,9iso andanteiso acids yet isolated from a marine sponge. The new acid 10,13-dimethyltetradecanoic (3), the unusual acid 15-methyl-11-hexadecenoic (4) and the also novel acid 9-methyl-11-hexadecenoic (5) were also identified inE. ferox. The principal sterols isolated fromE. ferox were 24-ethylcholest-5-en-3β-ol (46%) and 24(R)-methylcholesta-5,22-dien-3β-ol (14%).     

The sterols ofCucurbita moschata (“calabacita”) seed oil

From the sterol fraction of seed oil from commercialCucurbita moschata Dutch (“calabacita”) Δ⁵ and Δ⁷ sterols having saturated and unsaturated side chain were isolated by chromatographic procedures and characterized by spectroscopic (¹H and¹³C-nuclear magnetic resonance, mass spectrometry) methods. The main components were identified as 24S-ethyl 5α-cholesta-7,22E-dien-3β-ol (α-spinasterol); 24S-ethyl 5α-cholesta-7,22E, 25-trien-3β-ol (25-dehydrochondrillasterol); 24S-ethyl 5α-cholesta-7, 25-dien-3β-ol; 24R-ethylcholesta-7-en-3β-ol (Δ⁷-stigmastenol) and 24-ethyl-cholesta-7, 24(28)-dien-3β-ol (Δ^7,24(28)-stigmastadienol).Lipids 31, 1205–1208 (1996).     

Sterols ofKalanchoe pinnata: First report of the isolation of both C-24 epimers of 24-alkyl-Δ25-sterols from a higher plant

Eighteen sterols were isolated from the aerial parts ofKalanchoe pinnata (Crassulaceae) including four novel sterols,viz. (24R)-stigmasta-5,25-dien-3β-ol (24-epiclerosterol), (24R)-5α-stigmasta-7,25-dien-3β-ol, 5α-stigmast-24-en-3β-ol, and 25-methyl-5α-ergost-24(28)-en-3β-ol. 24-Epiclerosterol and its Δ⁷-analog occur together with their 24S/β-epimers in the same plant making this the first report of the isolation of both C-24 epimers of Δ²⁵-unsaturated 24-alkylsterols from a non-marine organism. Iodine-catalyzed isomerization of stigmasta-5,24-dien-3β-ol (24-ethyldesmosterol), the main sterol ofK. pinnata, yielded 24-epiclerosterol among other products.     

Anatomical distribution of sterols in oysters (Crassostrea gigas)

Oysters (Crassostrea gigas) contain at least 8 predominant sterols as determined by gas liquid chromatography and a modified Liebermann-Burchard reaction. These sterols and the average amount found in mg/100 are: C₂₆-sterol (22-trans-24-norcholesta-5, 22-diene-3β-ol), 19.1; 22-dehydrocholesterol, 15.1; cholesterol, 46.8; brassicasterol, 27.2; Δ^5,7-sterols (i.e., 7-dehydrocholesterol) 22.5; 24-methylenecholesterol 29.1; 24-ethylcholesta-5,22-diene-3β-ol, 1.2; and 24-ethylcholesta-5-en-3β-ol, 12.7. The distribution of these sterols appears uniform (r²=0.938) between 5 major organs of the oyster. The percent body mass vs percent total sterols in these 5 organs are: mantle 44.1–41.4; visceral mass 30.3–36.7; gills 13.2–11.7; adductor muscle 8.3–3.7; and labial palps 4.2–6.5. The possible sources of these sterols are discussed.     

Sterols of the spongeTethya amamensis: Occurrence of (24E)-24-ethylidenecholesta-5,7-dienol, (24E)-24-propylidenecholesta-5,7-dienol,and (24Z)-24-propylidenecholesta-5,7-dienol

The spongeTethya amamensis, collected from Kagoshima Bay, Japan, contained at least 24 sterols, including Δ⁵-sterols (82.2% of total sterols) and Δ^{5, 7}-sterols (17.8%). The predominant sterols were cholesterol (29.0%), cholesta-5,22-dienol (13.8%), 24-methylcholesta-5,22-dienol (10.9%), 24-methylenecholesterol (8.3%), 24-methylcholesta-5,7,22-trienol (6.8%), 24-ethylcholest-5-enol (6.1%), and isofucosterol *4.1%). Combined gas liquid chromatography-mass spectrometry suggested the presence of 3 uncommon sterols, (24E)-24-ethylidenecholesta-5,7-dienol, (24E)-24-propylidenecholesta-5,7-dienol, and (24Z)-24-propylidenecholesta-5,7-dienol as minor components. The sterols ofT. amamensis also contained small amounts of 24-norcholesta-5,7,22-trienol and (24Z)-24-ethylidenecholesta-5,7-dienol.     

Inhibition of sterol biosynthesis in chlorella ellipsoidea by AY-99441

WhenChlorella ellipsoidea was grown in the presence of 4 ppm AY-9944, complete inhibition of Δ⁵-sterol biosynthesis was achieved. However total sterol production remained unaltered. As a result a number of sterols accumulated that appear to be intermediates in sterol biosynthesis. These sterols were described and identified as (24S)-5α-ergost-8(9)-en3β-ol, (24S)-5α-stigmast-8(9)-en-3β-ol, 4α-methyl-(24S)-5α-ergosta-8, 14-dien-3β-ol, 4α-methyl-(24S)-5α-stigmasta-8, 14-dien-3β-ol, 4α-methyl-(24S)-5α-ergost-8(9)-en-3β-ol and (24S)-4α-methyl-5α-stigmast-8(9)-en-3β-ol. The occurrence of these sterols inChlorella ellipsoidea is the first time they have been noted in biological material. The accumulation of these sterols in treated cultures indicates that AY-9944 is an extremely effective inhibitor of the Δ⁸→Δ⁷ isomerase and the Δ¹⁴ reductase of these plants. The occurrence of small amounts of other sterols in treated cultures has led to a proposed pathway for thebiosynthesis of sterols inChlorella ellipsoidea. Scientific Article No. A1775, Contribution No. 4565 of the Maryland Agricultural Experiment Station.     

Detailed sterol compositions of two pathogenic rust fungi

Teliospores of cedar-apple rust Gymnosporangium juniperi-virginianae were collected from the eastern red cedar Juniperus virginiana, and aeciospores of quince rust G. clavipes were collected from the fruit of English hawthorn Crataegus laevigata. The sterol fractions were separated by HPLC, and their identities were determined by 600 MHz ¹H NMR. Twenty-six sterols were isolated from G. juniperi-virginianae and 18 sterols were isolated from G. clavipes. The principal sterol of both fungi was (Z)-stigmasta-7,24(28)-dien-3β-ol. Other major sterols were (24S)-ergost-7-en-3β-ol, (24S)-stigmast-7-en-3β-ol, and (24S)-stigmasta-5,7-dien-3β-ol. The sterols of the hosts were found to be very different from those of the fungi. The 24-alkyl sterols of the fungi had the 24α-configuration, whereas those of the hosts had the 24β-configuration. Similarities to the sterol composition of the AIDS pneumonia fungus Pneumocystis carinii are discussed.     

Characterization of sterols in refined borage oil by GC-MS

Borage oil sterols were isolated by TLC and characterized using GC and GC-MS. Several diunsaturated Δ⁵-sterols, some of them not previously recorded in vegetable oils, were found. Of these, 24-methylcholesta-5,23-dienol and 24-ethylcholesta-5,23-dienol could be useful markers for borage oil. Two other diunsaturated Δ⁵-sterols that are rarely found in vegetable oils, 24-methylcholesta-5,24(25)-dienol and 24-ethylcholesta-5,24(25)-dienol, were identified. The diunsaturated C-24(28)-sterol, isofucosterol, was also found, as well as the monounsaturated Δ⁵-sterols campesterol and sitosterol. These are normally present in vegetable oils, which makes them unsuitable as markers for borage oil.     

Sterol metabolism in the nematodeCaenorhabditis elegans

The metabolism of various dietary sterols and the effects of an azasteroid on sitosterol metabolism in the free-living nematodeCaenorhabditis elegans was investigated. The major unesterified sterols ofC. elegans in media supplemented with sitosterol, cholesterol or desmosterol included 7-dehydrocholesterol (66.5%, 40.5%, 31.2%, respectively), cholesterol (6.7%, 52.3%, 26.9%), lathosterol (4.4%, 3.6%, 1.7%) and 4α-methylcholest-8(14)-en-3β-ol (4.2%, 2.1%, 3.8%). Esterified sterols, representing less than 20% of the total sterols, were somewhat similar except for a significantly higher relative content of 4α-methylcholest-8(14)-en-3β-ol (23.3%, 23.4%, 10.6%). ThusC. elegans not only removes the substituent at C24 of dietary sitosterol but possesses the unusual ability to produce significant quantities of 4α-methylsterols. WhenC. elegans was propagated in medium supplemented with sitosterol plus 5 μg/ml of 25-azacoprostane hydrochloride, the azasteroid strongly interfered with reproduction and motility ofC. elegans and strongly inhibited the Δ24-sterol reductase enzyme system; excluding sitosterol, the major free sterols of azacoprostane-treatedC. elegans were cholesta-5, 7, 24-trien-3β-ol (47.9%), desmosterol (9.4%), fucosterol (2.1%) and cholesta-7,24-dien-3β-ol (2.0%). These 4 sterols are likely intermediates in the metabolism of sitosterol inC. elegans.     

Unusual tetraene sterols in some phytoplankton

Sterols were analyzed from four phytoplankton strains which are under investigation as possible sources of food for oysters in culture. One strain ofPyramimonas contained only 24-methylenecholesterol as a major sterol component.Pyramimonas grossii, Chlorella autotrophica andDunaliella tertiolecta each contained a complex mixture of C₂₈ and C₂₉ sterols with Δ⁷, Δ^5,7 and Δ^5,7,9(11) nuclear double bond systems. Sterols were found both with and without the C-22 side chain double bond. Ergosterol and 7-dehydroporiferasterol were the principal sterols in each of the latter three species, which also contained the rare tetraene sterols, 24-methylcholesta-5,7,9(11),22-tetraen-3β-ol and 24-ethylcholesta-5,7,9(11),22-tetraen-3β-ol.