Unknown Sterol Research Articles

Sterol profiling of Leishmania parasites using a new HPLC-tandem mass spectrometry-based method and antifungal azoles as chemical probes reveals a key intermediate sterol that supports a branched ergosterol biosynthetic pathway

Human leishmaniasis is an infectious disease caused by Leishmania protozoan parasites. Current chemotherapeutic options against the deadly disease have significant limitations. The ergosterol biosynthetic pathway has been identified as a drug target in Leishmania. However, remarkable differences in the efficacy of antifungal azoles that inhibit ergosterol biosynthesis have been reported for the treatment of leishmaniasis. To better understand the sterol biosynthetic pathway in Leishmania and elucidate the mechanism underlying the differential efficacy of antifungal azoles, we developed a new LC-MS/MS method to study sterol profiles in promastigotes of three Leishmania species, including two L. donovani, one L. major and one L. tarentolae strains. A combination of distinct precursor ion masses and LC retention times allowed for specific detection of sixteen intermediate sterols between lanosterol and ergosterol using the newly developed LC-MS/MS method. Although both posaconazole and fluconazole are known inhibitors of fungal lanosterol 14α-demethylase (CYP51), only posaconazole led to a substantial accumulation of lanosterol in azole-treated L. donovani promastigotes. Furthermore, a key intermediate sterol accumulated by 40- and 7-fold when these parasites were treated with posaconazole and fluconazole, respectively, which was determined as 4α,14α-dimethylzymosterol by high resolution mass spectrometry and NMR spectroscopy. The identification of 4α,14α-dimethylzymosterol supports a branched ergosterol biosynthetic pathway in Leishmania, where lanosterol C4- and C14-demethylation reactions occur in parallel rather than sequentially. Our results suggest that selective inhibition of leishmanial CYP51 is insufficient to effectively prevent parasite growth and dual inhibitors of both CYP51 and the unknown sterol C4-demethylase may be required for optimal antiparasitic effect.

11-β-hydroxysterols as possible endogenous stimulators of mitochondrial biogenesis as inferred from epicatechin molecular mimicry

Currently, there is great interest in identifying endogenous (i.e. physiological) stimulators of mitochondrial biogenesis (MB), in particular, those that may mediate the effects of exercise. The molecular size of the cacao flavanols (epicatechin and catechin) highly resembles that of sterols and epicatechin has been reported to activate cells surface receptors leading to the stimulation of MB in endothelial and skeletal muscle cells translating into enhanced exercise capacity. We therefore hypothesize, that epicatechin may be acting as a structural mimic of an as yet unknown sterol capable of stimulating MB. We developed a new synthetic process for obtaining enantiomerically pure preparations of (-)-epicatechin and (+)-epicatechin. Applying spatial analytics and molecular modeling, we found that the two isoforms of epicatechin, (-) and (+), have a structural resemblance to 11-β-hydroxypregnenolone, a sterol with no previously described biological activity. As reported in this proof-of-concept study performed in primary cultures of endothelial and muscle cells, 11-β-hydroxypregnenolone is one of the most potent inducers of MB as significant activity can be detected at femtomolar levels. The relative potency of (-)/(+)-epicatechin isoforms and on inducing MB correlates with their degree of spatial homology towards the 11-β-hydroxypregnenolone. On the basis of these results, the detailed in vivo characterization of the potential for these sterols to act as endogenous modulators of MB is warranted.

Structural Analysis of Smoothened Reveals a Sterol Binding Site

A previously unknown sterol binding pocket was found in the crystal structure of active Smoothened.

Fully automated determination of the sterol composition and total content in edible oils and fats by online liquid chromatography–gas chromatography–flame ionization detection

Sterol analysis of edible oils and fats is important in authenticity control. The gas chromatographic determination of the sterol distribution and total content is described by ISO norm 12228. Extraction, purification, and detection of the sterols are time-consuming and error-prone. Collaborative trials prove this regularly. Purification by thin-layer chromatography (TLC) and robust GC determination of all mentioned sterols is not straightforward. Therefore, a fully automated LC-GC-FID method was developed to facilitate the determination of sterols. The only manual step left was to weigh the sample into an autosampler vial. Saponification and extraction were performed by an autosampler while purification, separation, and detection were accomplished by online coupled normal-phase LC-GC-FID. Interlacing of sample preparation and analysis allowed an average sample throughput of one sample per hour. The obtained quantitative results were fully comparable with the ISO method with one apparent exception. In the case of sunflower oils, an additional unknown sterol was detected generally missed by ISO 12228. The reason was found in the omission of sterol silylation before subjection to GC-FID. The derivatization reaction changed the retention time and hid this compound behind a major sterol. The compound could be identified as 14-methyl fecosterol. Its structure was elucidated by GC–MS and ensured by HPLC and GC retention times. Finally, validation of the designed method confirmed its suitability for routine environments.

Sterol Composition and Biosynthetic Genes of Vitrella brassicaformis, a Recently Discovered Chromerid: Comparison to Chromera velia and Phylogenetic Relationship with Apicomplexan Parasites.

Vitrella brassicaformis is the second discovered species in the Chromerida, and first in the family Vitrellaceae. Chromera velia, the first discovered species, forms an independent photosynthetic lineage with V. brassicaformis, and both are closely related to peridinin-containing dinoflagellates and nonphotosynthetic apicomplexans; both also show phylogenetic closeness with red algal plastids. We have utilized gas chromatography/mass spectrometry to identify two free sterols, 24-ethylcholest-5-en-3β-ol, and a minor unknown sterol which appeared to be a C(28:4) compound. We have also used RNA Seq analysis to identify seven genes found in the nonmevalonate/methylerythritol pathway (MEP) for sterol biosynthesis. Subsequent genome analysis of V. brassicaformis showed the presence of two mevalonate (MVA) pathway genes, though the genes were not observed in the transcriptome analysis. Transcripts from four genes (dxr, ispf, ispd, and idi) were selected and translated into proteins to study the phylogenetic relationship of sterol biosynthesis in V. brassicaformis and C. velia to other groups of algae and apicomplexans. On the basis of our genomic and transcriptomic analyses, we hypothesize that the MEP pathway was the primary pathway that apicomplexans used for sterol biosynthesis before they lost their sterol biosynthesis ability, although contribution of the MVA pathway cannot be discounted.

Biochemical Characterization of Plasma Membrane Vesicles of Cyanophora paradoxa*

Abstract:Plasma membrane vesicles (PMVs) were isolated from Cyanophora paradoxa Korshikov (Claucocystophyta) and purified by differential and sucrose density gradient centrifugation. PMVs were identified ultrastructurally and biochemically using marker enzymes. Analysis of monosaccharides revealed a sugar content of 37.3% of dry weight of delipidated PMVs with high amounts of fucose (28 mol% of total carbohydrate), galactose (23%), mannose (21%) and the minor constituents xylose, rhamnose, glucose and N‐acetylglucosamine. Proteins were analyzed by SDS‐PACE. More than 20 polypeptides were resolved and several were identified as glycopeptides, as shown by PAS staining and lectin blots. Fucose linked α‐1→6 to N‐acetylglucosamine (AAA‐binding), mannose linked α‐1→2,3,6 to mannose (CNA‐binding) and terminally bound galactose (RCA‐binding) were identified on several polypeptides. Sterols (β‐sitosterol and an additional unknown sterol, 40% of total lipids) and diacylglycerol (43%) are the major lipid components in the PMVs of C. paradoxa. Minor components are PE, PC, PC and an unknown glycolipid. Fatty acid analysis revealed 12 different fatty acids, predominantly 16:0,18:0 and the polyunsaturated acids 20:4 and 20:5 (together 69% of total fatty acids), α‐Linolenic acid, common in higher plant PMs, could only be detected as a minor constituent (< 1 %) of PMVs of C. paradoxa.

Identification of 19-nor-5,7,9(10)-cholestatrien-3 beta-ol in patients with Smith-Lemli-Opitz syndrome

We have identified the third unknown sterol in the plasma and tissues of Smith-Lemli-Opitz homozygotes as 19-nor-5,7,9(10)-cholestatrien-3 beta-ol. The structure was established from capillary gas-liquid chromatography retention index and characteristic fragmentation pattern by mass spectrometry that were identical to a synthetic reference standard. Evidence is presented that 19-nor-5,7,9(10)-cholestatrien-3 beta-ol is not an artifact formed during the chemical isolation of the relatively unstable 7-dehydrocholesterol. It is possible that 19-nor-5,7,9(10)-cholestatrien-3 beta-ol may contribute to the clinical abnormalities in patients with Smith-Lemli-Opitz syndrome.

STEROL DISTRIBUTION IN THE GENUS HETEROCAPSA (PYRRHOPHYTA)1

ABSTRACTThe sterol composition of seven strains of marine peridinioid dinoflagellates comprising the four known species of Heterocapsa Stein was examined by gas chromatography‐mass spectrometry to determine the utility of these compounds in systematics. Cholest‐5‐en‐3β‐ol (cholesterol), 24‐methyl‐cholest‐5‐en‐3β‐ol (24‐methylcholesterol), 4α,24(S)‐dimethyl‐5α‐cholestan‐3β‐ol (4,24‐dimethylcholestanol), 4α,23,24(R)‐trimethyl‐5α‐cholest‐22‐en‐3β‐ol (dinosterol), 4α,23ξ,24ξ‐trimethyl‐5α‐cholestan‐3β‐ol (dihydrodinosterol), and an unknown sterol were detected. Sterol composition does not vary significantly from species to species within the genus Heterocapsa and thus cannot be used for species differentiation. Sterols may, however, have value in defining the properties of dinoflagellate taxa above the family level. Over the course of the growth curve for Heterocapsa niei (Loeblich) Morrill & Loeblich 4,24‐dimethylcholestanol and dinosterol covaried, suggesting that 4,24‐dimethylcholestanol is converted into dinosterol by a previously proposed bioalkylation scheme.

Identification of C-24 alkyl epimers of marine sterols by 13C nuclear magnetic resonance spectroscopy

13C nuclear magnetic resonance spectra of diastereomeric C-24 alkyl sterols have been assigned. Differences in the chemical shifts of side-chain carbons permitted the determination of the absolute configuration at C-24 in several sterols since these chemical shifts are insensitive to structural changes remote from the asymmetric centre. An unknown sterol from Tetraselmissuecica has been identified as (24R)-24-methylcholest-5-en-3β-ol and the configuration assigned from 1H nmr data to the sterol from Phaeodoctylumtricornutum has been confirmed. The utility and potential of this method in characterising new sterols and their biological precursors is discussed.

Study on the chemical nature of sterols contained in Bulgarian sunflower oil.

The free sterols, the sterol esters and the sterol glycosides of the crude sunflower oil as well as those of the technical lecithin, the pitch and the deodorizer distillate of the latter oil were isolated by preparative TLC. The nature of sterols contained in the isolated sterol derivatives was elucidated by GLC and combinded GLC-MS. Major sterols of all examined sterol fractions are sitosterol, campesterol and stigmasterol, the amount of sitosterol being prevalent. Unknown sterol with a molecular weight of 428 is present in sterols of the crude oil and the deodorizer distillate. Sterols of the deodorizer distillate contain an unknown sterol with a molecular weight of 430. Stigmasterol is present in the sterol fraction of the deodorizer distillate in high amounts. It was established that delta7-sterols of the crude oil occur only in esterified form. The hydrolysis of the sterol derivatives in acid medium leads to dehydration products known as steroid dienes and disteroid ethers. Hydrolysis without dehydration was achieved by enzyme preparations. Some sterols of the crude oil were esterified with the same higher fatty acids contained in the glycerides of the sunflower oil.

Sterol composition of Bulgarian soya and corn oils.

The free sterols, the sterol esters and the sterol glycosides of the raw soya and corn oils as well as those of the technical lecithin and the deodorizer distillated of the latter oils were isolated by preparative TLC. The composition of each of the isolated sterol derivatives was determined by GLC and MS. Sitosterol, campesterol, stigmasterol and an unknown sterol with a molecular weight of 428 are contained in almost all of the examined fractions of the soya oil and its refinement byproducts. Dehydrocampesterol is present in the free sterols of the raw soya oil and the soya lecithin. Stigmasterol is contained in the soya deodorizer distillate in high amounts. It was established that cholesterol was present in the sterol esters of the raw soya oil high amounts. Delta7-stigmastenol occurs only in the sterol esters of the latter oil. Sitosterol, campesterol and stimgasterol are the main components of all sterol fractions of the corn oil and its refinement products. Dehydrocampesterol and unknown sterols with molecular weights of 428 are present in the free sterols of the raw corn oil. Some sterol glycosides of the soya and corn lecithin are esterified with the same major fatty acid components of the glycerides, palmitic acid being the main one. The fatty acid compositon of sterol esters of the raw soya and corn oil roughly corresponds to the fatty acid composition of oils.

Sterols of healthy and rust-infected primary leaves of wheat and of non-germinated and germinated uredospores of wheat stem rust

Sterols extracted from healthy and rust-infected primary leaves of wheat or from non-germinated and germinated uredospores of stem rust were analyzed by thin-layer, column, and gas–liquid chromatography.Wheat leaves of susceptible and resistant lines contained cholesterol, campesterol, stigmasterol, β-sitosterol, and an unknown sterol that accounted for 75% to 80% of total sterol content. A further sterol, stigmast-7-enol, occurred in all rust-infected leaves and in 1 of 11 samples of healthy leaves. Sterol levels were not related to susceptibility or resistance. The level of stigmast-7-enol in infected leaves appeared to be correlated with the amount of fungal mass in the host.Uredospores of stem rust contained trace amounts of an unknown sterol, cholesterol, and either ergost-7-enol or stigmasterol, and larger amounts of stigmast-7-enol. After germination, the level of cholesterol increased, especially in differentiating uredosporelings. The level of stigmast-7-enol tended to decrease after germination.Sterol fractions from wheat leaves (healthy and rust-infected) and from stem rust uredospores (non-germinated and germinated) were not phytotoxic. They did not inhibit rust development in susceptible leaves nor did they promote it in resistant leaves.

Intermediates from dealkylation at C 24 of beta-sitosterol by the cricket Locusta migratoria L

The locustLocusta migratoria L. transformsβ-sitosterol into cholesterol. Fucosterol, desmosterol and an unknown sterol (probably in C29) are found to be intermediates, but not 24-methylene cholesterol.

Simultaneous spectrometric determination of 5-ene and 7-ene sterols from the leaves and seeds of Digitalis purpurea L.

A method is described for the simultaneous spectrophotometric determination of the 5–ene and the 7–ene sterols in extracts from the leaves and seeds of Digitalis purpurea L. The method is based on the observation that, when the sterols are mixed with the Liebermann–Burchard reagent, 7–ene sterols develop a colour more rapidly than the 5–ene sterols. A non–polar lipid fraction and a polar glycoside fraction were isolated from both the seeds and the leaves. In each fraction the 5–ene sterols were calculated with reference to 24α–ethylcholest–5–en–3β–ol (β–sitosterol), and the 7–ene type with reference to 24β–methylcholesta–5–7–22–trien–3β–ol (ergosterol). The mean error in the determination of the former was 2.7 % and for the latter was 4.5 %. By means of GLC the 5–ene sterols of the non–polar fraction were found to be cholesterol, an unknown sterol, campesterol, stigmasterol and β–sitosterol; and of the polar fraction, campesterol, cholesterol, stigmasterol and β–sitosterol.

アルテミアにおけるステロールの利用と生合成

In the present study, the utilization and biosynthesis of sterols in Artemia salina were investigated. The results obtained are as follows: 1) In spite of being supplied with the two types of diets containing ergosterol, stig-masterol, β-sitosterol, campesterol, and unknown sterol, Artemia was found to contain only cholesterol as a sterol. The result suggested that Artemia may modify these sterols to chole-sterol. 2) It was shown by the tracer experiment that Artemia did not incorporate acetate into sterols. The result indicated that Artemia may lack sterol-synthesizing ability.

Inhibitors of cholesterol synthesis and myelin formation.

AbstractInhibitors of cholesterol biosynthesis AY 9944 and 20,25 diazacholesterol were administered by stomach tube to suckling rats in varying doses during the time of rapid myelination (15–22 days of age). Purified myelin was prepared from the brains and spinal cords, and the sterol content analyzed. Up to 50% of the myelin sterol consisted of desmosterol in rats treated with 20,25 diazacholesterol, while 7‐dehydrocholesterol comprised at least one third of the myelin sterol in rats administered AY 9944. Myelin from rats treated with both compounds contained desmosterol, 7‐dehydrocholesterol, Δ5,7,24 cholestatriene‐3‐β‐ol and an unknown sterol, the four comprising about 45% of the total sterol. The proportion of phospholipid: galactolipid‐total sterol in myelin from the drug‐treated rats was not significantly different from the normal, although much less myelin was recovered. Brain and spinal cord slices from 22 to 25‐day rats pretreated with inhibitors showed decreased uptake of U‐14C‐glucose into all myelin components. The decreased uptake was approximately proportional in all lipids and the protein was also affected. It is proposed that myelin composition is fixed, and that a deficiency of one of the lipid components will limit the assembly of the whole lipid portion of the membrane.

Triterpene alcohols and sterols of vegetable oils

AbstractTriterpene alcohols and sterols were separated by thin‐layer chromatography and gas‐liquid chromatography from the unsaponifiable fractions of the following 18 vegetable oils: linseed, peanut, olive, rice bran, palm kernel, corn, sesame, oiticica, palm, coconut, rapeseed, grape seed, sunflower, poppy seed, castor, tea seed, cocoa butter and soybean. Two triterpene alcohols, cycloartenol and 24‐methylene cycloartanol, were found in all of the oils except soybean oil, which contained only cycloartenol. Triterpene alcohols such as ॅ‐ and ॆ‐amyrin, euphorbol, butyrospermol and cyclolaudenol also were encountered occasionally. Three sterols, ॆ‐sitosterol, stigmasterol and campesterol were present in all of the oils. In addition a fourth sterol, not yet idenfified, was found in oils of palm, palm kernel and sunflower in varying amounts. This unknown sterol and brassicasterol were found in rapeseed oil in addition to the three sterols that were common to all of the oils studied.

Sterine und triterpenoide—V : Über die 4α-methyl-sterine der kartoffelpflanze Solanum tuberosum L.

In addition to cycloartenol, sigmasterol, β-sitosterol, campesterol and cholesterol a mixture of 4α-monomethyl sterols has been isolated from leaves of the potato plant, Solanum tuberosum L., which is probably identicla with α 1-sitosterol. The structure of the main component of this mixture has been established as 4α-methyl-5α-stigmasta-7,24(28)-diene-3β-ol (24-ethylidene-lophenol, III). Molecular mass spectrographic and gas chromatographic investigations indicated in addition to III two minor 4α-methyl sterols, lophenol (I) and a yet unknown sterol, the structure of which was tentatively assumed to be 24-methylene-lophenol (II).

Studies on the Sterol of Bombyx mori L.

In whole stages of Bombyx silkworm it was shown with gas-liquid chromatographic systems that silkworm-sterols consist of three sterols such as cholesterol, β-sitosterol, and campesterol, at least, and the sterols in the 5th instar larva contain afore-mentioned three sterols and an additional unknown sterol. Moreover, the sterol composition of the silkworm was studied in various stages.

サイカチの不鹸化物について

A dried legume of Gleditschia horrida Makino (Leguminosae) is known to contain saponin but no studies have been made as to its non-saponifiable matter. Experiments by the present author resulted in isolation of various substances. Oil of Gleditschia horrida is a wax-like substance of dark greenish color, obtained in 1.2% yield. It gives an acid value of 127.6, saponification value of 228.8, iodine value of 87.9, contains 0.7% of sterol (by digitonin method) and 64.8% of non-saponifiable mater. The latter yielded ceryl alcohol, m.p. 78°, nonacosane, m.p. 63-64°, stigmasterol, sitosterol, and a structurally unknown sterol of m.p. 158-159°. Besides the foregoing, minute amount of substances assumed to be sitostene, heptacosane, and terpene-like matter were isoleted. Majority of the solid non-saponifiable matter were ceryl alcohol and nonacosane.