Phytosterol biosynthesis pathway in Mortierella alpina

The Zygomycetes fungus Mortierella alpina was cultured to growth arrest to assess the phytosterol biosynthesis pathway in a less-advanced fungus. The mycelium was found to produce 13 sterols, but no ergosterol. The sterol fractions were purified to homogeneity by HPLC and their identifies determined by a combination of GC-MS and 1H NMR spectroscopy. The principal sterol of the mycelium was cholesta-5, 24-dienol (desmosterol) (83%), with lesser amounts of 24beta-methyl-cholesta-5,25(27)-dienol (codisterol) (2%), 24-methyldesmosterol (6%), 24(28)-methylene cholesterol (3%) and lanosterol (3%) and several other minor compounds (3%). The total sterol accounted for approximately 0.07% of the mycelial dry wt. Mycelium fed methionine-methyl-2H3 for 6 days, generated 3 2H-24-methyl(ene) sterols, [C28-2H2]24(28)-methylenecholesterol, [C28-2H3]24-methylcholesta-5,24-dienol and [C28-2H3]24beta-methyl-cholesta-5,25(27)-dienol. The formation of the 24-methyl sterols seems to be catalyzed by the direct methylation of a common Delta24-acceptor sterol thereby bypassing the intermediacy of an isomerization step for rearrangement of the Delta24(28)-bond to Delta25(25)-position as operates in Ascomycetes fungi and all plants.     

Sterol Composition and Ecdysteroid Content of Eggs of the Root-knot Nematodes Meloidogyne incognita and M. arenaria

Free and esterified sterols of eggs of the root-knot nematodes Meloidogyne incognita races 2 and 3 and M. arenaria race 1 were isolated and identified by gas-liquid chromatography-mass spectrometry. The major sterols of eggs of each race were 24-ethylcholesterol (33.4-38.8% of total sterol), 24-ethylcholestanol (18.3-25.3%), 24-methylcholesterol (8.6-11.7%), 24-methylcholestanol (7.7-12.5%), and cholesterol (4.6-11.6%). Consequently, the major metabolic transformation performed by Meloidogyne females or eggs upon host sterols appeared to be saturation of the sterol nucleus. The free and esterified sterols of the same race did not differ appreciably, except for a slight enrichment of the steryl esters in cholesterol. Although the sterol composition of Meloidogyne eggs differed from that of other life stages of other genera of plant-parasitic nematodes, the three Meloidogyne races could not be distinguished from each other by their egg sterols. Ecdysteroids, compounds with hormonal function in insects, were not detected by radioimmunoassay in the Meloidogyne eggs either as free ecdysteroids or as polar conjugates.     

Sterols of the marine sponge Petrosia weinbergi: implications for the absolute configurations of the antiviral orthoesterols and weinbersterols

The marine sponge Petrosia weinbergi was found to contain isofucosterol and clionasterol as its major sterols. The rare cyclopropyl sterol (24S,28S)-24,28-methylenestigmast-5-en-3beta-ol, previously detected as only 0.07% of the total sterols of a pelagophytic alga Pulvinaria sp., made up 6.6% of the total sterols. These sterols are believed to be the biosynthetic precursors of the antiviral orthoesterols and weinbersterols found in the same sponge. Based on the side chains of the isolated sterols, the absolute configurations of the antiviral steroid side chains are assigned to be (24R,28S)- for orthoesterol B, (24R)- for orthoesterol C, and (24S,28S)- for weinbersterols A and B.     

Marine sterols. VI(1). Sterols of the scallop, Patinopecten yessoensis.

The sterols of the scallop, Patinopecten yessoensis Jay, was found to contain over 20 components. The major components were delta5-sterols, and lesser amount of ring-saturated sterols were also present. Biogenetically unusual C26 sterols (24-norcholesta-5,22-dien-3beta-ol and 24-norcholest-22-en-3beta-ol) and 24(28)-cis-24-propylidenecholest-5-en-3beta-ol (29-methylisofucosterol), 22-trans-27-nor-(24S)-24-methylcholesta-5,22-dien-3beta-ol (occelasterol), and a new sterol, 22-trans-27-nor-(24S)-24-methylcholest-22-en-3beta-ol (patinosterol), were isolated and their structures were confirmed. Occurrence of 22-trans-(24S)-24-methylcholesta-5,22-dien-3beta-ol (24-epibrassicasterol) was confirmed. 22-cis-Cholesta-5,22-dien-3beta-ol was not found.     

Effects of the hypocholesteremic agent trifluperidol on the sterol, steryl ester, and fatty acid metabolism of Saccharomyces cerevisiae.

Trifluperidol (TFP), at a concentration of 100 muM, inhibited the 24-h growth of Saccharomyces cerevisiae by about 30%. Effects on lipid metabolism were investigated by monitoring the incorporation of [1-14C]sodium acetate into various lipid fractions after 4 and 24 h of growth in the presence of several concentrations of TFP. Although little effect was noted on the amount of free sterols, 24-h incorporation of label into steryl esters was increased two- to fourfold by 100 muM TFP. Major sterol components of the steryl ester fraction isolated from an untreated culture were zymosterol (48%) and ergosterol (24%), whereas from the TFP-treated culture delta8,24(28)-ergostadienol (66.6%) and delta8-ergostenol (14.7%) were most abundant. Free sterols present in the highest concentration in the untreated culture were ergosterol (78.2%) and lanosterol (13%); whereas delta8,22-ergostadienol (38.5%), delta8-ergostenol (35.4%), and delta8,24(28)-ergostadienol (25.4%) were the most abundant free sterols obtained from the TFP-treated culture. Thus, the major block in the sterol biosynthetic pathway in yeast appears to be delta8 leads to delta7 isomerization. In these same cultures the relative amounts of C12 and C14 acids isolated from both steryl ester and miscellaneous lipid fractions were increased more than threefold over controls.     

Sterol Composition of the Corn Cyst Nematode,Heterodera zeae,and Corn Roots

Sterols from free sterol and steryl ester fractions from Heterodera zeae and from total lipids of Zea mays roots were analyzed by gas-liquid chromatography (GLC) and by GLC-mass spectrometry. The major free sterols of H. zeae were 24-ethylcholesterol (54.4% of total free sterol), 24-ethylcholesta-5,22-dien-3β-ol (13.3%), 24-methylcholesterol (12.5%), and cholesterol (7.2%). The same four sterols comprised 34.6%, 7.2%, 30.3%, and 18.6%, respectively, of the esterified sterols of H. zeae. Corn root sterols included 46.6% 24-ethylcholesta-5,22-dien-3β-ol, 16.7% methylcholesterol, 16.4% cycloartenol, 12.7% 24-ethylcholesterol, and 0.5% cholesterol. The sterol 24-composition of H. zeae differed greatly from that of the only other cyst nematode previously investigated, Globodera solanacearum.     

Ophirasterol, a new C31 sterol from the marine sponge Topsentia ophiraphidites

Analysis of the sterol fraction obtained from the Colombian Caribbean sponge Topsentia ophiraphidites revealed that this sponge is a rich source of C30 and C31 sterols. Among them, a new C31 sterol, named ophirasterol, was isolated, and its structure was established as (22E,24R)-24-(1-buten-2-yl)cholesta-5,22-dien-3beta-ol (1) by spectral means and comparison with synthetic C-24 epimers with known configuration. Other isolated C30 and C31 sterols were the known 24-ethyl-24-methyl-22-dehydrocholesterol (2), 24-isopropyl-22-dehydrocholesterol (3), 24-isopropylcholesterol (4), 24-ethyl-24-methylcholesterol (5), 24-isopropenyl-25-methyl-22-dehydrocholesterol (6) and 24-isopropenyl-25-methylcholesterol (7), and 24-isopropenyl-22-dehydrocholesterol (8).     

A simplified micro-method for quantification of fecal excretion of neutral and acidic sterols for outpatient studies in humans.

A simple and precise micro-method for measurement of daily fecal excretion of neutral and acidic sterols has been developed which utilizes sitostanol (24-ethyl-5 alpha-cholestane-3 beta-ol) as fecal flow and recovery marker. Extractions of sterols were performed from 50 microliters of fecal homogenate (feces-water 1:1), and analyses of neutral and acidic sterols were carried out by gas-liquid chromatography. The method is sensitive, precise, and easy to perform; the intra-assay variability yielded coefficients of variations of 1.9% and 3.5% (n = 6) for neutral and acidic sterols, respectively. The results from this method were compared with those obtained with the standard fecal flow marker chromic oxide. The correlation coefficients between the two markers were compared in 16 subjects and were 0.938 and 0.998 for excretion of neutral sterols and acidic sterols, respectively. Comparison of the fecal excretion of neutral and acidic sterols in 12 subjects determined from frozen samples and aliquots (approximately 1 g) sent by ordinary mail to the laboratory (transport time 1 to 5 days) gave identical results using sitostanol as fecal flow marker (818 +/- (SEM) 85 mg/day vs. 838 +/- 89 mg/day for neutral sterols and 417 +/- 59 mg/day vs. 414 +/- 60 mg/day for acidic sterols). The new micro-method is ideally suited for research laboratories in need of a simple, accurate, inexpensive, and high through-put method for measuring daily fecal excretion of neutral and acidic sterols, as well as total cholesterol synthesis, and can be performed on an outpatient basis.     

Metabolism of Δ0-, Δ5-, and Δ7-Sterols by Larvae of Heliothis zea

Heliothis zea was reared on artificial diets containing Δ⁵-sterols (cholesterol, campesterol, or sitosterol), Δ⁷-sterols (lathosterol, epifungisterol, or spinasterol), or Δ⁰-sterols (cholestanol, epicoprostanol, campestanol, or sitostanol) in order to determine how different dietary sterols affect the type of sterols present in the tissues of the late-sixth-instar larva. Although all of the dietary sterols (except epicoprostanol) supported the growth of the larvae, not all of the sterols were metabolized to the same end products. In each case, at least 80% of the sterols in the tissues of the larvae retained the same nucleus as that of the dietary sterol, indicating that H. zea carries out very little metabolism of ring B of Δ⁵-, Δ⁷-, and Δ⁰-sterols. The larvae dealkylated the Δ⁵-, Δ⁷-, and Δ⁰-alkylsterols to 24-desalkylsterols, but a greater percentage of the Δ⁵-alkylsterols were metabolized in this manner. The sterols present as free sterols in the larva were also present as esterifed sterols which accounted for 2–4% of the total sterols. Therefore, the sterol composition of the tissues of H. zea can be altered by varying the dietary sterols.     

Stereochemical aspects of the biosynthesis of the side chain of 9beta, 19-cyclopropyl sterols in maize seedlings treated with tridemorph

9β, 19-Cyclopropyl sterols such as 24-methyl pollinastanol accumulate dramatically in maize (Zea mays L. var LG 11) seedlings treated with Tridemorph (2,6-dimethyl-N-tridecyl-morpholine), a systemic fungicide (M. Bladocha, P. Benveniste, Plant Physiol 1983 41: 756-762). In contrast to the situation in control plants where 24-ethyl sterols predominate largely, 24-methyl sterols were more than 98% of total cyclopropyl sterols. In addition, 24-methyl cyclopropyl sterols were a mixture of (24-R)- and (24-S)-24-methyl epimers and are similar in that respect to the 24-methyl cholesterol of control plants. The presence of two epimers at C-24 has been previously explained by the operation of two routes (M. Zakelj, L. J. Goad, Phytochemistry 1983 22: 1931-1936). One may proceed via Δ²⁴⁽²⁸⁾- and Δ²⁴⁽²⁵⁾-sterols to produce the (24-R)-24-methyl epimer. The other route may involve reduction of either a Δ²⁴⁽²⁸⁾-, a Δ²³-, or a Δ²⁵-sterol intermediate to give the (24-S)-24-methyl epimer. Such intermediates have been searched for in excised Zea mays axes grown aseptically in the presence of Tridemorph and either [5-¹⁴C]mevalonic acid, or [Me-¹⁴C]-l-methionine. Whereas Δ²⁴⁽²⁸⁾- and Δ²⁴⁽²⁵⁾-cyclopropyl sterols were found in relatively large amounts, only traces of radioactivity were associated with Δ²⁵-sterols. Gas chromatography/mass spectrometry analysis of the sterols from axes grown in the presence of [Me-²H₃]-l-methionine showed that Δ²⁴⁽²⁸⁾-cyclopropyl sterols contained only two ²H atoms at C-28 as expected and that the 24-methyl pollinastanol fraction contained species with two ²H atoms and no species with three ²H atoms. These results indicate that both (24-R)- and (24-S)-epimers originate from a common Δ²⁴⁽²⁸⁾ precursor. After incubation of the axis with [5-¹⁴C,(4-R)-4-³H₁]mevalonic acid, the 24-methyl pollinastanol had a ³H:¹⁴C atomic ratio of 4:6 which is consistent with the intermediacy of a Δ²⁴⁽²⁵⁾-sterol. All these data are in accordance with a pathway where Δ²⁴⁽²⁸⁾-cyclopropyl sterols are isomerized to give Δ²⁴⁽²⁵⁾-cyclopropyl sterols which in turn would be reduced nonregiospecifically to yield both (24-R)- and (24-S)-24-methyl pollinastanols. A plausible mechanism for the reduction step is discussed.     

The use of the Dhcr7 knockout mouse to accurately determine the origin of fetal sterols

Mice with a targeted mutation of 3beta-hydroxysterol Delta(7)-reductase (Dhcr7) that cannot convert 7-dehydrocholesterol to cholesterol were used to identify the origin of fetal sterols. Because their heterozygous mothers synthesize cholesterol normally, virtually all sterols found in a Dhcr7 knockout fetus having a Delta(7) or a Delta(8) double bond must have been synthesized by the fetus itself but any cholesterol had to have come from the mother. Early in gestation, most fetal sterols were of maternal origin, but at approximately E13-14, in situ synthesis became increasingly important, and by birth, 55-60% of liver and lung sterols had been made by the fetus. In contrast, at E10-11, upon formation of the blood-brain barrier, the brain rapidly became the source of almost all of its own sterols (90% at birth). New, rapid, de novo sterol synthesis in brain was confirmed by the observation that concentrations of C24,25-unsaturated sterols were low in the brains of all very young fetuses but increased rapidly beginning at approximately E11-12. Reduced activity of sterol C24,25-reductase (Dhcr24) in brain, suggested by the abundance of C24,25-unsaturated compounds, seems to be the result of suppressed Dhcr24 expression. The early fetal brain also appears to conserve cholesterol by keeping cholesterol 24-hydroxylase expression low until approximately E18.     

Seasonal and geographical variations of sterol composition in snow crab hepatopancreas and pelagic fish viscera from Eastern Quebec

Sterol composition was determined in snow crab hepatopancreas and mackerel and herring viscera for various locations and collection periods. A simple and valuable method, using direct saponification and extraction with water-cyclohexane has been optimized to recover total sterol. They were identified and quantified as trimethylsilyl ether derivatives by GC-MS analysis. Method validation indicated excellent sensitivity (limit of quantification: 1.25 mg/100 g wet basis for cholesterol and desmosterol; 0.03-0.05 mg/100 g for other sterols), good reproducibility (CV%: 1.5-6.8) and accuracy (recovery%: 94-107). In crab hepatopancreas, cholesterol was the main sterol (67-76%), followed by desmosterol (19-24%). Phytosterols and molluscan sterols were also present in low quantity. A lower total sterol content with different composition was found in crabs from Magdalen Islands compared to those from Gaspé Peninsula or North Shore of the St-Lawrence Gulf. No seasonal variation was observed between collection periods, which were probably too close. Mackerel and herring viscera contained the same sterols as crab except for campesterol and sitosterol, but the cholesterol proportion was higher (93-98%). The higher abundance of sterols in herring caught in September vs. May would be related to an increase of the body lipid content during the summer.     

Three new 7-keto sterols from the Mediterranean sponge Clathrina clathrus

The CHCl3 extract of the Mediterranean sponge Clathrina clathrus was shown to contain three new sterols: (22 E)-3 beta-hydroxycholesta-5,8,22-trien-7-one (1), (22 E, 24 xi)-3 beta-hydroxy-24-methylcholesta-5,8,22-trien-7-one (2), and (22 E, 24 xi)-3 beta-hydroxy-24-ethylcholesta-5,8,22-trien-7-one (3), whose structures were elucidated on the basis of physico-chemical evidence.     

In vivo studies of sterol and squalene secretion by human skin

This work was aimed at studying the quantity and composition of sterols and squalene secreted by the human skin. Lipids secreted by the entire skin were recovered by Soxhlet extraction of the clothing worn by a patient for 24 hr with a chloroform-methanol azeotrope and by extracting the water of a shower taken by the patient at the end of the 24-hr period. Squalene and sterols were quantified by gas-liquid chromatography. Plant sterols were separated from total sterols by thin-layer chromatography. Free and esterified cholesterol were separated by digitonin precipitation. In eight adults, seven of them with hyperlipoproteinemia, the total skin secretion of cholesterol ranged from 59 to 108 mg/day, with a mean of 88 +/- 17 (SD) mg/day. There was no difference in cholesterol secretion between the normocholesterolemic individual and the hypercholesterolemic ones, nor were there any differences according to type of hyperlipoproteinemia. Free cholesterol amounted to 54 +/- 5% of the total cholesterol. The secretion of squalene ranged from 125 to 475 mg/day in five patients. The secretion of both squalene and cholesterol was quite constant for any individual on a given diet. Cholesterol constituted 95.6 +/- 0.5% of the digitonin-precipitable total body surface sterols of eight patients, and lathosterol, the next largest fraction, 3.4 +/- 0.4%. Total plant sterols formed only 0.65 +/- 0.38% and beta-sitosterol 0.35 +/- 0.23% of the skin surface sterols in six patients whose dietary beta-sitosterol intake ranged from 230 to 3400 mg/day.     

Sterol and Squalene Content of a Docosahexaenoic-Acid-Producing Thraustochytrid: Influence of Culture Age, Temperature, and Dissolved Oxygen

Thraustochytrid strain ACEM 6063, rich in omega-3 polyunsaturated fatty acids, was cultured at 15°C and 20°C in high (>40%) and low (<5%) dissolved oxygen (DO), and at 25°C in low-DO media. Samples were taken 4, 2, and 0 days before each culture reached peak biomass (T₋₄, T₋₂, and T_p, respectively). Twenty sterols, 13 of which were identified, were detected. Predominant were cholest-5-en-3β-ol, 24-ethylcholesta-5,22E-dien-3β-ol, 24-methylcholesta-5,22E-dien-3β-ol, and 2 coeluting sterols, one of which was 24-ethylcholesta-5,7,22-trien-3β-ol. These 4 sterols comprised 50% to 90% of total sterols. Cultures grown at high DO had simpler sterol profiles than those grown at low DO. Only the 4 sterols mentioned above were present at more than 3% of total sterols in high-DO cultures. In low-DO cultures, up to 6 additional sterols were present at more than 3% of total sterols. Culture age, temperature, and DO influenced squalene and sterol content. Total sterols (as a proportion of total lipids) decreased with increasing culture age. If organisms such as ACEM 6063 are to be used for commercial production of lipid products for human consumption, both their sterol content and factors influencing sterol production need to be characterized thoroughly. Received January 8, 2001; accepted March 6, 2001.     

Sterol-mediated regulation of mevalonic acid synthesis. Accumulation of 4-carboxysterols as the predominant sterols synthesized in a Chinese hamster ovary cell cholesterol auxotroph (mutant 215)

Chinese hamster ovary-215 (CHO-215) mutant cells are auxotrophic for cholesterol. Berry and Chang (Berry, D. J., and Chang, T. Y. (1982) Biochemistry 21, 573-580) suggested that the metabolic lesion was at the level of 4-methyl sterol oxidation. However, the observed cellular accumulation of lanosterol was not consistent with a defect at this metabolic site. With the use of a novel Silica Sep Pak sterol separation procedure, we demonstrated that 60-80% of the acetonesoluble lipid radioactivity in [5-3H]mevalonate-labeled CHO-215 cells was incorporated into acidic sterols. 7(8),Cholesten-4 beta-methyl,4 alpha-carboxy,3 beta-ol was the dominant end product. In addition to this acidic sterol, 7(8),24-cholestadien,4 beta-methyl,4 alpha-carboxy,3 beta-ol and 7(8),24-cholestadien,4 alpha-carboxy,3 beta-ol were also isolated. Incubation of cell-free extracts with [3H]7(8)-cholesten-4 beta-methyl, 4 alpha-carboxy,3 beta-ol and pyridine nucleotides confirmed that CHO-215 4-carboxysterol decarboxylase activity was less than 1% of that for wild type cells. Thus, a correspondence between decreased 4-carboxysterol decarboxylase activity and the spectrum of accumulated sterol products by intact CHO-215 cells was demonstrated. No detectable cholesterol was synthesized by CHO-215 cells. 3H-Product accumulation studies demonstrated that 7(8),24-cholestadien, 4 beta-methyl,4 alpha-carboxy,3 beta-ol increased prior to its subsequent saturation at the delta 24 carbon. Furthermore, the steady state ratio for delta 24-saturated acidic sterols/unsaturated acidic sterols was dependent on media cholesterol source and amount. Finally, the accumulated acidic sterol(s) were not regulatory signal molecules for the modulation of 3-hydroxy-3-methyl-glutaryl coenzyme. A reductase activity in response to cholesterol availability.     

Intracellular distribution and origin of sterols in Calendula officinalis leaves

In Calendula officinalis leaves 66% of all steryl forms are present in the ‘microsomal fraction’ (IV), 24% in the mitochondrial and Golgi membranes (III), 5% in the ‘chloroplast’ (II), 4% in the ‘cell wall and membrane’ (I) fraction and 1%. in the cytosol. Free sterols, their esters, glycosides and acylated glycosides are present in varying proportions in all cellular subtractions. Mevalonate-[2¹⁴C] labelling of sterols derived from various steryl forms showed that free sterols and all their derivatives, i.e. steryl esters and glucosides, are formed in fraction IV and are then translocated to other organelles. Fraction III is the main site of glycosylation of transported sterols as well as of acylation of steryl glycosides.     

Sterols in blood of normal and Smith-Lemli-Opitz subjects

Smith-Lemli-Opitz syndrome (SLOS) is a hereditary disorder in which a defective gene encoding 7-dehydrocholesterol reductase causes the accumulation of noncholesterol sterols, such as 7- and 8-dehydrocholesterol. Using rigorous analytical methods in conjunction with a large collection of authentic standards, we unequivocally identified numerous noncholesterol sterols in 6 normal and 17 SLOS blood samples. Plasma or erythrocytes were saponified under oxygen-free conditions, followed by multiple chromatographic separations. Individual sterols were identified and quantitated by high performance liquid chromatography (HPLC), Ag(+)-HPLC, gas chromatography (GC), GC-mass spectrometry, and nuclear magnetic resonance. As a percentage of total sterol content, the major C(27) sterols observed in the SLOS blood samples were cholesterol (12;-98%), 7-dehydrocholesterol (0.4;-44%), 8-dehydrocholesterol (0.5;-22%), and cholesta-5,7,9(11)-trien-3beta-ol (0.02;-5%), whereas the normal blood samples contained <0.03% each of the three noncholesterol sterols. SLOS and normal blood contained similar amounts of lathosterol (0.05;-0.6%) and cholestanol (0.1;-0.4%) and approximately 0.003;-0.1% each of the Delta(8), Delta(8(14)), Delta(5,8(14)), Delta(5,24), Delta(6,8), Delta(6,8(14)), and Delta(7,24) sterols.The results are consistent with the hypothesis that the Delta(8(14)) sterol is an intermediate of cholesterol synthesis and indicate the existence of undescribed aberrant pathways that may explain the formation of the Delta(5,7,9(11)) sterol. 19-Norcholesta-5,7,9-trien-3beta-ol was absent in both SLOS and normal blood, although it was routinely observed as a GC artifact in fractions containing 8-dehydrocholesterol. The overall findings advance the understanding of SLOS and provide a methodological model for studying other metabolic disorders of cholesterol synthesis.     

The neutral sterols of Megalotomus quinquespinosus (say) (hemiptera: Alydidae) and identification of makisterone a as the major free ecdysteroid

Last-stage nymphs of the broad-headed bug, Megalotomus quinquespinosus contain the C₂₈ ecdysteroid makisterone A as their major ecdysteroid. No ecdysone or 20-hydroxyecdysone was detected in whole body extracts analyzed by high performance liquid chromatography and radioimmune assay. Analyses of the neutral sterols of this phytophagous hemipteran revealed that the sterol composition of the nymphs was highly reflective of their dietary sterols. The most abundant nymphal sterols were sitosterol (46.6%), Δ⁷-stigmastenol (13.8%) and spinasterol (13.4%). Cholesterol accounted for only 0.2% of the total sterols and indicates that this species is incapable of converting 24-alkyl sterols to cholesterol.     

Manipulation by 25-azacycloartanol of the relative percentage of C10, C9 and C8 side-chain sterols in suspension cultures of bramble cells

The addition of 25-azacycloartanol to the medium of suspension cultures of bramble cells resulted, after 6 weeks of growth, in a large decrease in the percentage of C₁₀ side-chain sterols, sitosterol and isofucosterol (83 % of the total in the control, 9 % in the treated cells), and in a spectacular increase in the percentage of C₈ side-chain sterols, cycloartenol, desmosterol and cholesterol (less than 1 % in the control, 53 % in the treated cells). In addition the relative percentage of C₉ side-chain sterols, mainly 24-methylene cholesterol increased significantly (from 16 to 37 %). A secondary effect of 25-azacycloartanol consisted in an increase of the percentage of Δ²⁴ sterols and in a decrease of the percentage of sterols with a saturated side chain. These results are in agreement with an inhibition by 25-azacycloartanol of the C-24 and C-28 methyltransferases and of the Δ²⁴ reductase.