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.     

Reversed-phase thin layer chromatography of some common sterols

The chromatographic mobilities of 17 sterols and squalene on reversed-phase thin layer plates with four nonaqueous solvent systems is described. A degree of separation adequate to identify several of the sterols was obtained. It was possible to separate the pairs: cholestanol, epicholestanol; coprostanol, epicoprostanol; 5β-cholestan-3α and 3β-ol and lanosterol, dihydrolanosterol.     

Unusual composition of sterols in a phytophagous insect,Mexican bean beetle reared on soybean plants

Three saturated sterols, cholestanol, campestanol, and stigmastanol, constituted 54, 72, and 77% of the total sterols of the egg, prepupa, and adult, respectively, of the Mexican bean beetle,Epilachna varivestis (Mulsant), reared on soybean plants. Lathosterol (7-cholesten-3β-ol), possibly formed from cholestanol in this insect, constituted 12, 16, and 11.8% of the total sterols isolated from egg, prepupa, and adult, respectively. None of these sterols have been isolated and identified previously as components of the sterols of a phytophagous insect reared on a natural host plant. Cholesterol, a major sterol of most plant feeding insects studied thus far, comprised less than 5% of the total sterols in any of the stages examined. The unique composition of the sterols in this insect in relation to the sterol composition of the host plant is compared to dietary sterol utilization and metabolism in other phytophagous insects. ARS, USDA.     

Sterol content of some plant oils; Further observations on fast-reacting sterols

Total and free sterols were measured by a modified Sperry-Webb procedure in raw and refined corn, cottonseed, safflower seed, linseed, soybean and wheat germ oils. Wheat germ and safflower seed oil sterols were relatively rich in fastreacting sterols, which predominated in the sterol ester fraction. Photometric constants following color reaction were obtained for cholesterol and plant sterols and applied to a procedure for analysis of cholesterol and plant sterols in mixtures thereof.     

Role of sterols in membranes

Sterols, or in rare cases structurally similar molecules, are biosynthesized or at least required by all eucaryotic organisms, as well as by many procaryotic ones, regardless of their status as plants, animals, or protista. This information, together with quantitative, structural, metabolic, and other data is reviewed. It is interpreted to mean that the primary role sterols play in nature is a nonmetabolic one as architectural components of membranes and that this role can be played, but less well, by other molecules which approximate the steroidal structure. The biosynthetic process should, therefore, and actually does not appear to be correlatable with this role, which, in turn, is correlatable with phylogenesis. The Δ²⁴-reduction-alkylation bifurcation, for instance appears to be interrelated profoundly with the evolutionary differentiation of the animal from the plant kingdom. One of eight papers presented in the symposium “Phytosterols,” AOCS Spring Meeting, New Orleans, April 1973.     

Functions of sterols in plants

Sterols have at least three functions in animals: they may act as precursors of other steroids, as hormones and as membrane components. The author advances the hypothesis that sterols have similar functions in plants. One of 12 papers to be published from the “Sterol Symposium” presented at the AOCS Meeting, New Orleans, April 1970. W. Utiliz. Res. Dev. Div., ARS, USDA.     

The distribution of sterols in algae

Available analytical techniques are now sufficient for the separation and identification of sterols from complex mixtures in plants. Gas and thin layer chromatography and mass spectroscopy in particular, have been used to resolve some of the confusion concerning the sterol composition of algae. Red algae (Rhodophyta) contain primarily cholesterol, although several species contain large amounts of desmosterol, and one species contains primarily 22-dehydrocholesterol. Only a few Rhodophyta contain traces of C-28 and C-29 sterols. Fucosterol is the dominant sterol of brown algae (Phaeophyta), apparently the major sterol of every species examined. Most Phaeophyta also contain traces of cholesterol and biosynthetic precursors of fucosterol. The sterols of green algae (Chlorophyta) are much more varied and complex than those of other groups of algae. Whereas the Phaeophyta and Rhodophyta contain one primary sterol, many of the Chlorophyta contain a complex mixture of sterols such as occurs in higher plants. The Chlorophyta contain such sterols as chondrillasterol, poriferasterol, 28-isofucosterol, ergosterol, cholesterol and others. Sterol composition may be of value in the systematics of plants such as the Chlorophyta. Recently (for the first time) complex mixtures of sterols have been isolated in very small amounts in the blue-green algae (Cyanophyta). Available data on the sterols of other groups of algae are insufficient for making useful comparisons. One of 12 papers to be published from the “Sterol Symposium” presented at the AOCS Meeting, New Orleans, April 1970. Scientific Article No. A2606, Contribution No. 4331 of the Maryland Agricultural Experiment Station.     

Male-specific tetraene and triene hydrocarbons ofCarpophilus hemipterus: Structure and pheromonal activity

Males ofCarpophilus hemipterus (L.), the dried-fruit beetle, (Coleoptera: Nitidulidae) were found to emit nine all-E tetraene and one all-E triene hydrocarbons in addition to two pheromonally active tetraenes that had been reported previously. The previously known compounds are (2E,4E,6E,8E)-3,5,7-trimethyl-2,4,6,8-decatetraene(1) and (2E,4E,6E,8E)-3, 5,7-trimethyl-2,4,6,8-undecatetraene(2). The new tetraenes were all related to structure1 by having one additional carbon at either one or two of the following four locations: at carbon 1 of the chain, at carbon 10 of the chain, at the 5-alkyl branch, or at the 7-alkyl branch. (Structure 2 also fits within this pattern.) The triene inC. hemipterus is (2E,4E,6E)-5-ethyl-3-methyl-2, 4,6-nonatriene. Also identified from volatile collections from the beetles were the 2Z and 4Z isomers of1. All structures were proven by synthesis, with NMR and mass spectral data for the compounds provided. Two of the newly discovered compounds, (2E,4E,6E,8E)-7-ethyl-3,5-dimethyl-2,4,6,8-decatetraene and (2E,4E,6E,8E)-7-ethyl-3,5-dimethyl-2,4,6,8-undecatetraene, were quite active in the wind-tunnel bioassay, but others, such as (2E,4E,6E,8E)-5-ethyl-3,7-dimethyl-2,4,6,8-decatetraene and (2E,4E,6E,8E)-4,6,8-trimethyl-2,4,6,8-undecatetraene were not. Structureactivity relationships are explored among the natural compounds and additional, synthetic analogs, which were never detected from the beetles. Some of these analogs, such as (2E,4E,6E,8E)-3,5-dimethyl-7-propyl-2,4,6,8-undecatetraene, were quite active in the bioassay. The biosynthesis of the beetle-derived compounds is discussed. A single biosynthetic scheme that lacks complete enzyme specificity at four specific steps could account for the entire series of compounds found in the beetles and their relative proportions. The definition of pheromone is discussed in relation to these hydrocarbons.     

Grapefruit seed oil sterols

Grapefruit seed oil sterols separated from other lipids by Florisil column chromatography were characterized by gas liquid chromatography. The presence of stigmasterol, campesterol and β-sitosterol is indicated. Expressed in terms of peak area, the three sterols are present in proportions of 2.5%, 7.4% and 90.1% of the total, respectively.     

Cholestanol and plant sterols in pigeon skin

Cholestanol (4.6%) and plant sterols (0.2%) have been demonstrated, for the first time, in avian skin by argentation and gas liquid chromatography. In contrast to results of previous studies with rat and human skin, cholestanol represented a significant amount in the pigeon. The proportion of campesterol was always higher than that of β-sitosterol.     

The phylogenetic distribution of sterols in tracheophytes

The sterols of nine mature plant species in seven families ranging from the subphylum Lycopsida through the Filicopsida and the classes Gymnospermae and Angiospermae in the Pteropsida were structurally and stereochemically defined. Two plant categories were found. In the first, comprised byDryopteris (Thelypteris) noveboracensis, Polystichum acrostichoides, Dennstaedtia punctilobula, Osmunda cinnamomea, Ginkgo biloba, Cucurbita pepo, andKalmia latifolia, 24α-alkylsterols were dominant and were composed principally of 24α-ethylcholesterol (sitosterol) or (inCucurbita pepo) 24α-ethyllathosterol and itstrans-22-dehydro derivative (spinasterol). Depending on the species, small amounts of 24α-ethyl-trans-22-dehydrocholesterol (stigmasterol), 24α-methylcholesterol (campesterol), 24β-methylcholesterol (dihydrobrassicasterol, always less than campesterol), cholesterol, lathosterol, 24α-ethyllathosterol, 24ξ-methyllathosterol,trans-24-ethylidenelathosterol (Δ⁷-avenasterol), and (tentatively identified) 24-ethyl-24(25)-dehydrolathosterol were present.Spinacea oleracea was also confirmed as belonging to Category I, and, except as described in what follows, Category I represents all other configurationally investigated vascular plants. The second category of plants contained only 24β-ethylsterols. Only one species (Kalanchoe daigremontiana) belonging to the family Crassulaceae was found, but one other (the genusClerodendrum in the family Verbenaceae) is already known.K. daigremontiana contained 25(27)-dehydroclionasterol (clerosterol) and 25(27)-dehydroporiferasterol. The primitiveLycopodium complanatum was intermediate between Categories I and II; the sterols with a 24-C₂-group had only the 24α-configuration (sitosterol with some stigmasterol), but the principal sterols with a 24-C₁-group (ergosterol and dihydrobrassicasterol) possessed the 24β-configuration.C. pepo seeds, which are already known to contain principally 24β-ethylsterols, contrast sharply with our finding that tissue (pericarp of the fruit) from the mature plant contains only 24α-ethylsterols. This apparent evolutionary recapitulation (Category II to I) during development coupled with statistical dominance of Category II plants among the algae and fungi and of Category I plants in the Tracheophytes, and the existence of an intermediate type in the species examined from the lower Tracheophyte (Lycopsida) lead logically to the conclusion that the 24α-alkyl structure, especially 24-α-ethyl-Δ⁵-sterols (sitosterol and the much rarer stigmasterol) constitutes the most highly evolved type of 24-alkylsterol. By inference from our knowledge of biosynthesis, corroborated by the spectrum of sterols found here, the pathway [through Δ²⁴⁽²⁵⁾-sterol] leading to 24α-alkylsterol appears to be higher than the pathway [through Δ²⁵⁽²⁷⁾-sterols] which leads to 24β-alkylsterols. The sterols of these and other plants were also found amenable to classification according to their nuclear unsaturation (Δ⁵, A; Δ⁷, B; and Δ^5,7, C). Tracheophytes of Category A have been most frequently encountered, butC. pepo was shown to be of Category B throughout its ontogeny. While no Tracheophytes of pure Category C have been discovered,L. complanatum was shown to be of the mixed A-C-Type. Based on these facts and ideas, some previously suggested lines of botanical evolution are examined. The chemical data fail to verify a line from Magnoliales to theCucurbitaceae, from Magnoliales through Theales to Ericales, nor from Ranales to Saxifragales. However, they are consonant with a relationship between Cucurbitaceae and Theales and between Rosales and Lamiales. Triterpenoids found in various of the families studied included cycloartenol and friedelin. The spectroscopic properties of the latter are described.     

Occurrence of plant sterols in aquatic vertebrates

Plant sterols were found by gas liquid chromatography in the sterols of five species of aquatic vertebrates; mackerel (Scomber japonicus), rainbow trout (Salmo gairdnerii), smelt (Osmerus dentex), sardine (Sardinops melanosticta) and chimera (Chimera phantasma). The sterols of chimera liver, sardine flesh and sardine viscera contained 9.0, 2.4 and 3.1% of C₂₈ and C₂₉ sterols in addition to 86.7, 96.6 and 95.2% of cholesterol. The occurrence of norcholestandienol, campesterol, β-sitosterol and C₂₈ stanol was shown by combined gas chromatography-mass spectrometry. Sperm whale (Physeter catodon) sterols consisted of more than 99% cholesterol with only traces of 22-dehydrocholesterol.     

Pharmaceutical-grade sterols from tall oil

Summary A process has been developed for producing pharmaceutically-pure sterols from tall oil pitch. The process consists of the propane fractionation of the pitch, saponification of the overhead fraction in methanol, fractional crystallization, centrifuging, washing, and drying. A plant to produce 1,000 lbs. of sterols per day was brought into operation. This paper won first place in the 1958–59 Tall Oil Award of the Tall Oil Division of the Pulp Chemicals Association.     

银杏叶中甾醇的GC-MS分析

通过对银杏叶中的甾醇进行分离,重结晶纯化,得到了高纯度的植物甾醇.采用GC-MS联用技术对甾醇进行了分析.色谱条件:HP-5色谱柱(30 m×0.25 mm×0.25μm);载气为高纯度N2,体积流量1 mL/min;进样温度300℃;柱温285℃,每分钟升高5℃,320℃保持30 min;电离方式EI,电子能量70 eV;接口温度250℃;离子源温度200℃;检测电压350 V;进样量1μL.经GC-MS确定了其中丰度较大的5种物质及其结构,分别为菜油甾醇、豆甾-4,22-双烯-3β-醇、β-谷甾醇、豆甾-3β-醇和岩藻甾醇.该方法简便、结果可靠,可用于分析银杏叶中甾醇的化学成分.     

Unusual effects of some vegetable oils on the survival time of stroke-prone spontaneously hypertensive rats

Preliminary experiments have shown that a diet containing 10% rapeseed oil (low-erucic acid) markedly shortens the survival time of stroke-prone spontaneously hypertensive (SHRSP) rats under 1% NaCl loading as compared with diets containing perilla oil or soybean oil. High-oleate safflower oil and high-oleate sunflower oil were found to have survival time-shortening activities comparable to that of rapeseed oil; olive oil had slightly less activity. A mixture was made of soybean oil, perilla oil, and triolein partially purified from high-oleate sunflower oil to adjust the fatty acid composition to that of rapeseed oil. The survival time of this triolein/mixed oil group was between those of the rapessed oil and soybean oil groups. When 1% NaCl was replaced with tap water, the survival time was prolonged by ∼80%. Under these conditions, the rapeseed oil and evening primrose oil shortened the survival time by ∼40% as compared with n-3 fatty acid-rich perilla and fish oil; lard, soybean oil, and safflower oil with relatively high n-6/n-3 ratios shortened the survival time by roughly 10%. The observed unusual survival time-shortening activities of some vegetable oils (rapeseed, high-oleate safflower, high-oleate sunflower, olive, and evening primrose oil) may not be due to their unique fatty acid compositions, but these results suggest that these vegetable oils contain factor(s) which are detrimental to SHRSP rats.     

Metabolism of plant sterols by nematodes

Parasitic nematodes do not biosynthesize sterolsde novo and therefore possess a nutritional requirement for sterol, which must be obtained from their hosts. Consequently, the metabolism of phytosterols by plant-parasitic nematodes is an important process with potential for selective exploitation. The sterol compositions of several species of plant-parasitic nematodes were determined by capillary gas chromatography-mass spectrometry and compared with the sterol compositions of their hosts. Saturation of the phytosterol nucleus was the major metabolic transformation performed by the root-knot nematodesMeloidogyne arenaria andM. incognita and the corn root lesion nematode,Prytalenchus agilis. In addition to saturation, the corn cyst nematode,Heterodera zeae, dealkylated its host sterols at C-24. Because free-living nematodes can be cultured in sterol-defined artificial medium, they have been successfully used as model organisms for investigation of sterol metabolism in plant-parasitic nematodes. Major pathways of phytosterol metabolism inCaenorhabditis elegans, Turbatrix aceti andPanagrellus redivivus incleded C-24 dealkylation and 4α-methylation (a pathway unique to nematodes).C. elegans andT. aceti introduced double bonds at C-7, andT. aceti andP. redivivus saturated the sterol nucleus similarly to the plant-parasitic species examined. Several azasteroids and long-chain dimethylalkylamines inhibited growth and development ofC. elegans and also the Δ²⁴-sterol reductase enzyme system involved in the nematode C-24 dealkylation pathway. Based on a paper presented at the Symposium on Plant and Fungal Sterols: Biosynthesis, Metabolism and Function, heald at the AOCS Annual Meeting, Baltimore, MD, April 1990.     

Free and combined sterols ofPavlova gyrans

Pavlova gyrans, a unicellular alga of interest as food for oysters, was cultured axenically and examined for sterol composition. Desmethyl monohydroxy sterols, which are frequently seen in algae, made up 40% of the total sterols and were observed primarily in the free sterol fraction. The principal sterols of this group were 5-ergostenol, poriferasterol, and clionasterol, as well as some poriferast-22-enol and poriferastanol. Several “methyl” sterols with unusual structures made up 27% of the total sterols. The principal “methyl sterols” were 4α-methyl ergostanol, 4α-methyl poriferastanol, and 4α-methyl poriferast-22-enol. Methyl sterols were found primarily in the ester fraction. Also observed was a new class of dihydroxysterols composing 33% of the total sterols. These sterols are structurally related to the methyl and desmethyl sterols ofPavlova but contain an extra nuclear hydroxyl which can be acetylated when present on a desmethyl sterol, but which is nonreactive with acetic anhydride in 4α-methyl sterols. None of these sterols were observed in ester form but are concentrated in the acid-hydrolyzable, bound fraction. The unique nature of these sterols suggests potential taxonomic utility. Based on a paper presented at the Symposium on Plant and Fungal Sterols: Biosynthesis, Metabolism and Function, held at the AOCS Annual Meeting, Baltimore, MD, April 1990.     

The quantitative analysis of plant sterols

Two methods for the quantitative analysis of plant sterols have been described. In the first, cholesterol is used for control of recovery and gas liquid chromatography (GLC) analysis. When cholesterol is present in the sterol mixture, a radioactive standard (usually cholesterol or sitosterol) is used to control recovery, and coprosterol is used to monitor GLC. The methods are exemplified for nitrogenfixing root nodules and for chloroplasts, respectively.