Diversity of sterol biosynthetic capacity in the caryophyllidae

The order Caryophyllales, along with its two associated orders, the Polygonales and Plumbaginales, comprise the angiosperm subclass Caryophyllidae. We have now characterized the sterol compositon of 231 members of this subclass. This includes 210 species and 21 cultivars in 108 genera within the 14 families of these three orders. From these data, clear differences in biosynthetic capability and putative relationships between taxa have been established. Members of the two monofamilial orders (Polygonales and Plumbaginales) contain Δ⁵-sterols in ratios typical of “main line” angiosperms. Members of families in the Caryophyllales contain Δ⁵-sterols, or Δ⁷-sterols or mixtures of Δ⁵- and Δ⁷-sterols. In the majority of species where Δ⁷-sterols are the dominant sterols produced, trace amounts to almost equal amounts of Δ⁵-sterols are also present. Replicate samples of many of these species have shown that the ratio of Δ⁵-sterols to Δ⁷-sterols in these species is stable over time and/or location. From these data, it appears that the conversion of Δ⁷-sterols to Δ⁵-sterols is highly regulated in the majority of species within this order. In these families, similarities in sterol composition correlate well with taxonomic relatedness. Relationships between these taxa with respect to biosynthetic capability can now be postulated. Based on a paper presented at the Symposium on Plant and Fungal Sterols: Biosynthesis, Metabolism and Function, held at the AOCS Annual Meeting, Baltimore, Maryland, April 1990.     

Taxonomic implication of sterol composition in the genusChlorella

Thirty-five isolates of the genusChlorella were grown under standardized conditions and analyzed for sterol composition. Six different sterol synthetic patterns were found. Two patterns were characterized by nuclear unsaturation at C-5 and one type by nuclear unsaturation at C-7. The remaining isolates all synthesize sterols with a diunsaturated nucleus (C-5+C-7) with various modifications. Subtype 1 synthesizes only C₂₈ sterols, subtype 2 produces C₂₈ and C₂₉ sterols, and subtype 3 (1 isolate) produces C₂₈ sterols with nuclear unsaturation at C-5+C-8 in addition to the Δ^5,7 C₂₈ sterols seen in the first 2 subtypes. Comparison of the sterol composition of strains shared in common with taxonomic researchers showed good correlation with the taxonomic scheme of Fott and Novakova which has been confirmed by Kessler and coworkers using other biochemical markers. Scientific article no. A-3030, contribution no. 6093 of the Maryland Agricultural Experiment Station.     

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.     

Characterization and composition of sterols in the free and esterified sterol fractions ofAspergillus oryzae

Free and esterified sterol fractions were isolated fromAspergillus oryzae, and their components analyzed mainly by gas chromatography-mass spectrometry. Cholesterol, brassicasterol, episterol (5α-ergosta-7,24[28]-dien-3β-ol), 4α-methyl-5α-ergosta-8[9],24[28]-dien-3β-ol, lanosterol and 24-methylene-24-dihydrolanosterol were well characterized, whereas 5-dihydroergosterol, sitosterol and 24[28]-dehydroergosterol were tentatively characterized. The principal sterol of the free sterol fraction was brassicasterol, and that of the esterified sterol fraction was episterol. Ergosterol, which is reported to be widely distributed in the fungi kingdom, was not detected.     

cis-5-Monoenoic fatty acids in some chenopodiaceae seed oils

Methyl esters from seed oils of four Chenopodiaceae species are unusual in that they contain methylcis-5-hexadecenoate (4.6–12%) and methyl 5-octadecenoate (1.1–1.2%). There are indications of small amounts of 18∶2^5,9 and 18∶3^{5, 9, 12} along with unsaturated acids commonly found in seed oils-oleic (14–21%), linoleic (53–57%) and linolenic (3.5–7.8%). Fatty acid composition of the oils was determined by gas chromatography, and positions of the double bonds were established by application of gas chromatography-mass spectrometry to the methoxylated methyl esters. N. Market. Nutr. Res. Div., ARS, USDA.     

Analysis of sterol content and composition in fats and oils by capillary-gas liquid chromatography using an internal standard. Comments on the German sterol method

The advantages and disadvantages of various approaches to the capillary-gas liquid chromatography (GLC) of sterols in fats and oils with internal standards are discussed. The German method F-III 1²(,3) for sterol analysis in fats and oils, which uses betulin as an internal standard, was reformulated and translated. A number of further improvements and alterations were also made, and the procedure used is described more precisely below in English in order to give detailed explanations, e.g. regarding the role of the aluminium oxide column and of the internal standard betulin. The method is applicable to a wide range of oils and fats, including mixtures of vegetable and animal fats, cereal lipids, and partially hydrogenated fats.     

Effect of altered sterol composition on the osmotic behavior of sphaeroplasts and mitochondria ofSaccharomyces cerevisiae

The effect of sterols on the osmotic stability of mitochondrial and plasma membranes of yeast wild-types and mutants that are defective in ergosterol biosynthesis has been studied. Incorporation of the nonfungal sterol, cholesterol, into yeast membranes reduces membrane elasticity which is observed as an increased susceptibility to osmotic lysis. However, the wild-type and nystatin-resistant strains which were examined indicate that qualitative alterations in endogenously generated sterols do not affect resistance to swelling. Although these strains exhibit differences in membrane fluidity, which is influenced by the sterol accumulated by the organisms, the membrane stretching capacity shows no distinct dependence on sterol structure or bilayer fluidity.     

The content and composition of sterols and sterol esters in sunflower and poppy seed oils

The composition and proportion of free sterols and sterol esters in crude sunflower and poppy seed oils were determined, using preparative thin layer chromatography followed by gas chromatography with cholesterol as an internal standard. Free sterols and sterol esters were also isolated in a liquid fraction obtained by low temperature crystallization (−80 C) of the oils and enriched with minor lipid classes. This enrichment procedure provided a liquid fraction suitable for studies of minor components in the oils. However, selectivity towards sterol esters was observed since sterols esterified to very long chain fatty acids (C₂₀–C₂₄) were preferentially retained in the precipitate. The proportions of free and esterified sterols were found to be 0.34 and 0.28%, respectively, in the sunflower oil, whereas the corresponding figures for poppy seed oil were 0.33% and 0.05%. Sunflower oil was characterized by a relatively high percentage of Δ7-sterols, preferentially obtained in the esterified fraction, and by very long chain saturated fatty acids of sterol esters. The sterols in poppy seed oil were composed almost entirely of campesterol, stigmasterol, sitosterol and Δ5-avenasterol, although their percentage distributions were remarkably different in the free and esterified fraction.     

Differences in sterol composition of gonads of the lottiid limpets Nipponacmea concinna and Nipponacmea fuscoviridis from northeastern Japan

This is the first report on the sterol composition in Nipponacmea concinna and Nipponacmea fuscoviridis, 2 dominant species of lottiid limpets. There were significant differences in sterol composition between male and female gonads of the limpets. Previous studies have shown that zymostenol and zymosterol are major lipid components of male gonads of the nacellid limpets Cellana grata and Cellana toreuma. In contrast, in this study, only trace amounts of zymosterol were detected in male gonads of N. fuscoviridis.     

Fatty acid and sterol composition of ungerminated spores of the vesicular-arbuscular mycorrhizal fungus,Acaulospora laevis

The fatty acids and sterols of ungerminated chlamydospores of the vesicular-arbuscular (VA) endophyteAcaulospora laevis were examined by gas chromatography and mass spectrometry. The total lipid content of the spores was 45.5% of the spore dry weight. Predominant fatty acids were palmitoleic (52.5%), palmitic (25.5%) and oleic (7.4%). Minor fatty acids consisted of a range of (n−3) and (n−6) polyunsaturated acids. The occurrence of (n−3) polyunsaturated fatty acids is rare in fungi of the order Mucorales. Three sterols were identified as 24-ethylcholesterol (79.9%), cholesterol (11.0%) and 24-methylcholesterol (9.2%). No ergosterol was detected. Lipids of the chlamydospores ofA. laevis are compared with those ofGlomus caledonius.     

The Effect of processing on the content and composition of free sterols and sterol esters in soybean oil

The content and composition of free sterols and sterol esters in crude soybean oil and in oils from different stages of two continuous refining systems were determined. The sterols were isolated by preparative thin layer chromatography and analyzed by gas chromatography with cholesterol as an internal standard. The free sterols in one of the degummed oils amounted to 3.1 mg/g and were diminished to 1.8 mg/g oil by the De Laval Short-Mix refining process. The content of free sterols of the other degummed oil was reduced from 3.4 to 1.6 mg/g oil by the Zenith process. The greatest reduction of sterol content was caused by the treatment with bleaching earth. The sterol esters accounted for 0.6 mg/g of the degummed oil, and only very small changes were observed during the processes. However, changes in the composition of fatty acids of the sterol esters were found. These changes might indicate a selective deacylation of sterol esters or an interesterification during the refining processes. The composition of sterols in free and esterified form were different. Campesterol, stigmasterol and sitosterol were obtained in both free and esterified form, but Δ7 stigmasterol was only found in esterified form. Only small changes in the percentage distribution of the sterols occurred during the processes. Present adress Food Technology Division, ALFA-LAVAL,S-14700 Tumba, Sweden     

Effect of host sterols on the sterol composition and virulence of a nuclear polyhedrosis virus ofHeliothis zea

The type of sterol in the diet ofHeliothis zea affected not only the sterol composition of the insect larva but also the virulence and/or sterol composition of a single-nucleocapsid nuclear polyhedrosis virus (HzSNPV). This baculovirus, which was purified by differential and sucrose density gradient centrifugation, had a sterol content of 40 ng per 10⁶ polyhedra. When the sterol composition of HzSNPV was characterized by gas liquid chromatography, reversed phase-high performance liquid chromatography, mass spectrometry, proton nuclear magnetic resonance spectrometry and/or ultraviolet spectroscopy, the sterols in the virus were similar to those of the host. The HzSNPV isolated from larvae fed Δ_{5_}, Δ_{0_} or Δ_5,7-sterols contained primarily cholesterol, cholestanol or 7-dehydrocholesterol, respectively. Changes in the sterol composition of HzSNPV affected its LD₅₀, but not LT₅₀, in larvae containing Δ₅-sterols. The LD₅₀ of virus isolated from larvae containing Δ_{0_}, Δ_{5_} and Δ₇-sterols decreased from 275,423 to 32,359 to 5,012 polyhedra/larva, respectively. The latter virus was also more virulent than the one that was isolated from larvae containing Δ_5,7-sterol and had an LD₅₀ of 58,884 polyhedra/larva. In contrast, the LD₅₀ of an HzSNPV (Sandoz, Inc.) containing Δ₅-sterol was not affected by the presence of Δ_{5_}, Δ_{0_} or Δ_5,7-sterols in the tissues of the host (1,413; 1,288 and 355 polyhedra/larva, respectively). The results of this study indicate that the sterol composition ofH. zea can affect the sterol composition of HzSNPV and therefore may affect the ability of this biological control agent to control its economically important insect host.     

Regulation of the sequencing in sterol biosynthesis

The evolutionary development and sequencing of events in the biosynthetic pathway leading to sterols is reviewed and illustrated by recent experiments from the author's laboratory. One of 12 papers being published from the “Sterol Symposium” presented at the AOCS Meeting, New Orleans, April 1970.     

Fatty acid composition of the sterol ester fraction of human adrenal cortex in Cushing's syndrome and after treatment with aminoglutethimide

The lipid that accumulated in the adrenal cortex of a patient who had been treated with aminoglutethimide has been compared with the lipid in normal human adrenal cortex and identified as esters of cholesterol with fatty acids. While the concentration of free cholesterol was normal, that of esterified cholesterol was three times greater than that in normal controls. Sterols were analyzed by gas chromatography and found to consist almost wholly of cholesterol. The fatty acid composition of the cholesterol esters in adrenal cortex from patients with abnormal steroid secretion rates was determined. An increased proportion of cholesteryl arachidonate (cholesteryl-3β-[allcis]-eicosa-5,8,11,14-tetraenoate) was found in adrenal cortex from patients with decreased steroid secretion rates and a decreased proportion in adrenal cortex from patients with steroid secretion rates raised sufficiently to cause Cushing's Syndrome. Publication no. 1429 of the Cancer Commission of Harvard University.     

Lipid,sterol and fatty acid composition of antarctic krill (Euphausia superba Dana)

The lipid classes, fatty acids of total and individual lipids and sterols of Antarctic krill (Euphausia superba Dana) from two areas of the Antarctic Ocean were analyzed by thin layer chromatography (TLC), gas liquid chromatography (GLC) and gas liquid chromatography/mass spectrometry (GLC/MS). Basic differences in the lipid composition of krill from the Scotia Sea (caught in Dec. 1977) and krill from the Gerlache Strait (caught in Mar. 1981) were not observed. The main lipid classes found were: phosphatidylcholine (PC) (33–36%), phosphatidylethanolamine (PE) (5–6%), triacylglycerol (TG) (33–40%), free fatty acids (FFA) (8–16%) and sterols (1.4–1.7%). Wax esters and sterol esters were present only in traces. More than 50 fatty acids could be identified using GLC/MS, the major ones being 14∶0, 16∶0, 16∶1(n−7), 18∶1(n−9), 18∶1(n−7), 20∶5(n−3) and 22∶6(n−3). Phytanic acid was found in a concentration of 3% of total fatty acids. Short, medium-chain and hydroxy fatty acids (C≤10) were not detectable. The sterol fraction consisted of cholesterol, desmosterol and 22-dehydrocholesterol.     

The sterol composition of freshly harvested compared to stored seeds of rape,sunflower and poppy

The composition of the free sterols and the sterol esters of freshly harvested seeds of rape, sunflower and poppy was compared to that of stored seeds. The sterol composition of rapeseed was not changed during storage, whereas in sunflower seed the free sterols had less of Δ5-avenasterol and Δ7-stigmastenol in ten-month-old seeds compared to fresh seeds. The greatest relative changes were observed for esterified sterols in poppy seed, with a drop in the percentage of Δ5-avenasterol from 25.3% in freshly harvested to 16.9% in seeds stored for 10 months.     

The content and composition of sterols and sterol esters in low erucic acid rapeseed (Brassica napus)

The low temperature crystallization technique for the enrichment of “minor” components, such as sterols and sterol esters, from vegetable oils was applied to low erucic acid rapeseed oils. The recovery of free sterols and sterol esters was estimated by use of¹⁴C-cholesterol and¹⁴C-cholesterol oleate. 80% of the free sterols and 45% of the sterol esters were recovered in the liquid fraction, while in two studies total recoveries were 95% and 99%, respectively. This technique showed some selectivity toward the sterol bound fatty acids when compared to direct preparative thin layer chromatography (TLC) of the crude oil. Gas liquid chromatography (GLC) analysis of the free and esterified sterols as TMS-derivatives showed very little selectivity in the enrichment procedure. The fatty acid patterns of the sterol esters demonstrated, however, a preference in the liquid fraction for those sterol esters which have a high linoleic and linolenic acid content. The content of free sterols was 0.3–0.4% and that of sterol esters 0.7–1.2% of the rapeseed oils in both winter and summer types of low erucic acid rapeseed (Brassica napus) when the lipid classes were isolated by direct preparative TLC of the oils. The free sterols in the seven cultivars or breeding lines analyzed were composed of 44–55% sitosterol, 27–36% campesterol, 17–21% brassicasterol, and a trace of cholesterol. The esterified sterols were 47–57% sitosterol, 36–44% campesterol, 6–9% brassicasterol, and traces of cholesterol and Δ5-avenasterol. The fatty acid patterns of these esters were characterized by ca. 30% oleic acid and ca. 50% linoleic acid, whereas these acids constitute 60% and 20%, respectively, of the total fatty acids in the oil. Little or no variation in sterol and sterol ester patterns with locality within Sweden was observed for the one cultivar of summer rapeseed investigated by the low temperature crystallization technique.     

Effect of extraction system, stage of ripeness, and kneading temperature on the sterol composition of virgin olive oils

Comparative extraction trials were carried out among a classical pressing, a dual-, and a three-phase centrifugation system using olive crops of Koroneiki variety. Two different kneading temperatures, 30 and 45°C, were tested at three stages of ripeness for two consecutive years of harvest, 1995–1996 and 1996–1997. Composition of the sterol fraction was determined in the resulting olive oil samples (n=72). Stigmasterol was found to be affected by the extraction system; it was obtained in the highest amount in the pressing system. The ratio campesterol/stigmasterol was significantly higher in oils extracted by dual- and three-phase centrifugation. Sterols were significantly affected by the ripening stage of the fruit. During December, the ratio campesterol/stigmasterol reached the maximal and β-sitosterol the minimal values; this appears to be the optimal period for harvesting the olives. Comparison of the different kneading temperatures showed that at 30°C, Δ⁵-avenasterol and campesterol/stigmasterol ratio reached higher values than at 45°C.     

Neutral sterol metabolism in insects

Since they are unable to biosynthesize sterols, many phytophagous and omnivorous insects satisfy their cholesterol requirement by side chain dealkylation of the C-24 alkyl group of dietary C₂₈ and C₂₉ phytosterols. However, not all insects that can dealkylate the phytosterol side chain produce cholesterol. In addition, certain insects,e.g., some Hymenoptera, Hemiptera, and Diptera, are unable to dealkylate the sterol side chain. Although C₂₇ ecdysteroids (molting hormones), which are biosynthesized from cholesterol, are the major ecdysteroids in most insects, many of those species that are unable to dealkylate phytosterols utilize campesterol as a precursor for the C₂₈ ecdysteroid makisterone A. The considerable diversity of steroid utilization between certain insect species makes it difficult to generalize about insect steroid biochemistry. The ability to disrupt certain unique aspects of steroid utilization and metabolism in insects might be exploited for developing new insect control technology. 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.     

Effect of colestipol on sterol metabolism in the rat

Sterol metabolism studies using isotopic and chromatographic techniques were performed on rats fed diets supplemented with colestipol (Upjohn). Compared to controls, colestipol altered sterol metabolism dramatically. Bile acid output increased from 7.0 mg/day to 12.2 mg/day (0.42% colestipol) and 39.6 mg/day (1.67% colestipol). Daily fecal neutral sterol output and daily endogenous neutral sterol output increased 36% and 55%, respectively, on the 1.67% colestipol diet. Cholesterol absorption was reduced by colestipol feeding. Cholesterol balance increased dramatically with 1.67% colestipol administration (43.5 mg/day vs −1.0 mg/day in controls). Colestipol exerts its effect by binding bile acids and by bile acid depletion interfering with cholesterol absorption.