Dietary sterol preference in the silkworm, Bombyx mori

Since insects are unable to biosynthesize sterols de novo, sterols must be obtained from dietary sources. Although it has been reported that beta-sitosterol is crucial for larval growth in the silkworm, Bombyx mori, little has been investigated concerning the dietary selection of sterols by Bombyx larvae. Here, we demonstrate that Bombyx larvae have the following sterol preference: beta-sitosterol > ergosterol > cholesterol = stigmasterol. Interestingly, Bombyx larvae preferred ergosterol, an inhibitory sterol on larval growth, indicating that sterol selection following first contact of the diet with the mouth part might be different from the sterol recognition mechanism present in sterol metabolism.     

Dietary Sterol Preference in the Silkworm,Bombyx mori

Since insects are unable to biosynthesize sterols de novo, sterols must be obtained from dietary sources. Although it has been reported that β-sitosterol is crucial for larval growth in the silkworm, Bombyx mori, little has been investigated concerning the dietary selection of sterols by Bombyx larvae. Here, we demonstrate that Bombyx larvae have the following sterol preference: β-sitosterol >> ergosterol > cholesterol = stigmasterol. Interestingly, Bombyx larvae preferred ergosterol, an inhibitory sterol on larval growth, indicating that sterol selection following first contact of the diet with the mouthpart might be different from the sterol recognition mechanism present in sterol metabolism.     

Quantification of sterol lipids in plants by quadrupole time-of-flight mass spectrometry

Glycerolipids, sphingolipids, and sterol lipids constitute the major lipid classes in plants. Sterol lipids are composed of free and conjugated sterols, i.e., sterol esters, sterol glycosides, and acylated sterol glycosides. Sterol lipids play crucial roles during adaption to abiotic stresses and plant-pathogen interactions. Presently, no comprehensive method for sterol lipid quantification in plants is available. We used nanospray ionization quadrupole-time-of-flight mass spectrometry (Q-TOF MS) to resolve and identify the molecular species of all four sterol lipid classes from Arabidopsis thaliana. Free sterols were derivatized with chlorobetainyl chloride. Sterol esters, sterol glycosides, and acylated sterol glycosides were ionized as ammonium adducts. Quantification of molecular species was achieved in the positive mode after fragmentation in the presence of internal standards. The amounts of sterol lipids quantified by Q-TOF MS/MS were validated by comparison with results obtained with TLC/GC. Quantification of sterol lipids from leaves and roots of phosphate-deprived A. thaliana plants revealed changes in the amounts and molecular species composition. The Q-TOF method is far more sensitive than GC or HPLC. Therefore, Q-TOF MS/MS provides a comprehensive strategy for sterol lipid quantification that can be adapted to other tandem mass spectrometers.     

Dietary sterols/steroids and the generalist caterpillar Helicoverpa zea: Physiology,biochemistry and midgut gene expression

Sterols are essential nutrients for insects because, in contrast to mammals, no insect (or arthropod for that matter) can synthesize sterols de novo. Plant-feeding insects typically generate their sterols, commonly cholesterol, by metabolizing phytosterols. However, not all phytosterols are readily converted to cholesterol. In this study we examined, using artificial diets containing single sterols/steroids, how typical (cholesterol and stigmasterol) and atypical (cholestanol and cholestanone) sterols/steroids affect the performance of a generalist caterpillar (Helicoverpa zea). We also performed sterols/steroids analyses, using GC/MS techniques, to explore the metabolic fate of these different dietary sterols/steroids. Finally, we used a microarray approach to measure, and compare, midgut gene expression patterns that arise as a function of dietary sterols/steroids. In general, H. zea performed best on the cholesterol and stigmasterol diets, with cholesterol as the dominant tissue sterol on these two treatments. Compared to the cholesterol and stigmasterol diets, performance was reduced on the cholestanol and cholestanone diets; on these latter treatments stanols were the dominant tissue sterol. Finally, midgut gene expression patterns differed as a function of dietary sterol/steroid; using the cholesterol treatment as a reference, gene expression differences were smallest on stigmasterol, intermediate on cholestanol, and greatest on cholestanone. Inspection of our data revealed two broad insights. First, they identify a number of genes potentially involved in sterol/steroid metabolism and absorption. Second, they provide unique mechanistic insights into how variation in dietary sterol/steroid structure can affect insect herbivores.     

Distribution and dynamic state of sterols and steroids in the tissues of an insect, the roach Eurycotis floridana

The total concentrations of sterols in the tissues of the roach, Eurycotis floridana, reared under aseptic conditions and on semisynthetic diets, are similar to, but somewhat lower than, those of tissues of vertebrates. Total concentrations of tissue sterols are relatively independent of dietary concentration of sterols whether the diet contains 0.1% cholesterol as the sole sterol, or a "minimal cholesterol" mixture (0.1% cholestanol together with 0.005% cholesterol). Under the latter conditions the cholesterol is incorporated preferentially into most tissues and remains almost exclusively unesterified, while the cholesterol-sparing sterol is esterified to varying degree, depending upon the tissue. The turnover of tissue sterols has been studied. Cholesterol of the tissues of adult insects grown on a diet containing this sterol alone may be displaced by cholestanol fed as 5% of the total diet, initially at an appreciable rate but later much less rapidly. In growing insects that have received a diet containing cholestanol together with minimal cholesterol, the unesterified cholesterol turns over slowly in all tissues and immeasurably slowly in some. The unesterified sparing sterol, on the other hand, turns over at a much greater rate. The turnover of sterols during growth is accompanied by a shift of sterols from the unesterified to the esterified pool in all tissues. The fat body of the growing insect stores sterols (apparently as their esters) that have been displaced from other tissues. The fat body of the adult does not show evidence of sterol storage. Polar derivatives of sterols are present in minor amount in all tissues of the insect, most abundantly in the mid-intestine and gastric caeca. These compounds seem likely to be C(27) steroids.     

Highly sensitive analysis of sterol profiles in human serum by LC-ESI-MS/MS

We have developed a highly sensitive and specific method for the analysis of serum sterol profiles. Sterols in 1 mul of dried serum were derivatized into picolinyl esters (3beta-picolinate) and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using the electrospray ionization (ESI) mode. In addition to cholesterol, 19 cholesterol precursors, cholestanol, campesterol, sitosterol, and sitostanol were identified simultaneously. Quantitative analyses for the picolinyl esters of 11 available sterols were performed, and detection limits were found to be less than 1 pg on-column. Reproducibilities and recoveries of 8 noncholesterol sterols were validated according to one-way layout and polynomial equation, respectively. The variances between sample preparations and between measurements by this method were calculated to be 1.6% to 8.2% and 2.5% to 16.5%, respectively. The recovery experiments were performed using 1 mul aliquots of normal human serum spiked with 1 ng to 6 ng of sterols, and recoveries of the sterols ranged from 88.1% to 102.5% with a mean recovery of 98.1%. The present method provides reliable and reproducible results for the identification and quantification of neutral sterols, especially in small volumes of blood samples, which is useful for serological diagnosis of inherited disorders in cholesterol metabolism and for noninvasive evaluation of cholesterol biosynthesis and absorption in humans.     

Sterol composition in larvae of the silkworm, Bombyx mori

Sterols in silkworm larvae were analyzed. Cholesterol was predominantly detected in all tissues examined. Dietary phytosterols and desmosterol, a putative biosynthetic intermediate from phytosterols to cholesterol, were also detected, indicating that imperfect intestinal conversion from phytosterols to cholesterol influences the sterol composition in larval tissues.     

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.     

The role of ABC proteins Aus1p and Pdr11p in the uptake of external sterols in yeast: dehydroergosterol fluorescence study

Uptake of external sterols in the yeast Saccharomyces cerevisiae is a multistep process limited to anaerobiosis or heme deficiency. It includes crossing the cell wall, insertion of sterol molecules into plasma membrane and their internalization and integration into intracellular membranes. We applied the fluorescent ergosterol analog dehydroergosterol (DHE) to monitor the initial steps of sterol uptake by three independent approaches: fluorescence spectroscopy, fluorescence microscopy and sterol quantification by HPLC. Using specific fluorescence characteristics of DHE we showed that the entry of sterol molecules into plasma membrane is not spontaneous but requires assistance of two ABC (ATP-binding cassette) pumps – Aus1p or Pdr11p. DHE taken up by uptake-competent hem1ΔAUS1PDR11 cells could be directly visualized by UV-sensitive wide field fluorescence microscopy. HPLC analysis of sterols revealed significant amounts of exogenous ergosterol and DHE (but not cholesterol) associated with uptake-deficient hem1Δaus1Δpdr11Δ cells. Fluorescent sterol associated with these cells did not show the characteristic emission spectrum of membrane-integrated DHE. The amount of cell-associated DHE was significantly reduced after enzymatic removal of the cell wall. Our results demonstrate that the yeast cell wall is actively involved in binding and uptake of ergosterol-like sterols.     

Effect of cholesterol deficiency on the composition of phospholipids and fatty acids of the housefly, Musca domestica

The housefly larvae were grown in the aseptic diet containing 0.56 μmole cholesterol/g wet weight of diet (control) and 0.05 μmole cholesterol/g wet weight of diet (deficient). The effects of cholesterol deficiency upon the phospholipid composition and fatty acids of the total phospholipid and triglyceride fractions from the lipid extract of the various larval tissues, whole larva, and in both sexes of adults 4 days after eclosion were examined. The total sterol and phospholipid contents (expressed relative to the wet weight of the insect) of the control and deficient insects at the larval and adult stages were analysed and molar ratios compared. The results suggest that cholesterol deficiency reduced the free sterol content of the larvae and adult insects to approximately 25% of the content of the control insects. However, cholesterol deficiency did not effect the phospholipid content during larval and adult stages when compared to that of control insects. Though the larvae reared on the cholesterol deficient diets did not show a profound alteration in the phospholipid composition, a marked increase in the ratio of phosphatidylcholine to phosphatidylethanolamine of the larval fat body and composite gut fraction were noticed. The cholesterol deficiency induced significant changes in the fatty acid composition of the phospholipid fraction of the insect. The ratio of unsaturated fatty acids to saturated fatty acids of the phospholipid fractions decreased significantly due to cholesterol deficiency in the whole larvae and in both sexes of adult flies. The data indicates that cholesterol deficient insects compensated for the lack of cholesterol by increasing saturated fatty acids preferentially in the phospholipid fraction of the lipids for the maintenance of proper membrane fluidity.     

Validation of an isotope dilution gas chromatography-mass spectrometry method for analysis of 7-oxygenated campesterol and sitosterol in human serum

High dose daily intake of plant sterols decreases the uptake of cholesterol in the intestine by competitive mechanisms and thus leads to reduced serum levels of total and LDL-cholesterol. By this, the commercialization of plant sterol enriched ‘functional food’ products is rapidly increasing. Subjects using these kinds of diet present a duplication of their serum plant sterol levels after long-term intake. In analogy to cholesterol, plant sterols such as campesterol and sitosterol can be oxidized to oxyphytosterols and these may counteract the primary anti-atherosclerotic action of cholesterol lowering. In order to investigate the whole spectrum of the consequences following high plant sterol intake a highly sensitive and specific isotope dilution gas chromatography–mass spectrometry method for the analysis of 7-oxygenated campesterol/sitosterol in trace amounts in human serum is presented in this paper. The validation was based on limits for detection and quantification, recovery, precision and minimization of autoxidation during work-up. Our results show an overall coefficient of variation ≤10% for the precision. The lowest limits for detection and quantification for 7α-hydroxy-campesterol were 7 pg/mL and 23 pg/mL, respectively. Data for overall sum recovery ranged from 92% to 115%. We practically used this method for analysis of oxyphytosterols simultaneously with plant sterol concentrations in serum from healthy volunteers. Sixteen subjects were treated with plant sterol enriched margarine (3 g/day) for 28 days. The results showed a significant increase of the oxyphytosterol 7β-hydroxy-sitosterol from 1.19 ± 0.54 (before intake) to 2.24 ± 1.24 ng/mL (mean ± SD; +86.7%; P = 0.007) after intake of the margarine. There was a highly significant correlation between the serum levels of campesterol and the sum of 7-oxygenated campesterol (R² = 0.915; P < 0.001) and sitosterol and the sum of 7-oxygenated sitosterol (R² = 0.915; P < 0.001). We can conclude from this study that the analytic method is well suited for detection of OPS, even at trace amounts.     

Effects of diet and metamorphosis upon the sterol composition of the butterfly Morpho peleides

Whole body sterol metabolism in insects has seldom been studied. We were able to design an appropriate study at a butterfly farm in Belize. We collected six larvas of butterfly (Morpho peleides), their food (leaves of Pterocarpus bayessii), and their excretions. In addition, six adult butterflies were collected. The sterols of the diet, the larva, and adult butterfly were analyzed by gas-liquid chromatography. The structures of these sterols were identified by digitonin precipitation, GC-MS, and NMR. Four sterols (cholesterol, campesterol, stigmasterol, and sitosterol) and a sterol mixture were found in the food, the body, and the excreta of the larva. The tissue sterol content of the larva was 326 microg. They consumed 276 microg of sterols per day. Their excretion was 185 microg per day as sterols. The total tissue sterol contents of the larva and butterfly were similar, but they had different sterol compositions, which indicated interconversion of sterols during development. There was a progressive increase in the cholesterol content from larva to butterfly and a decrease in the content of sitosterol and other plant sterols, which were likely converted to cholesterol. Our data indicated an active sterol metabolism in butterfly larva. Diet played an important role in determining its sterol composition. During metamorphosis, there was an interconversion of sterols. This is the first paper documenting the fecal sterol excretion in insects as related to dietary intakes.     

A new method for the quantitation of propofol in human plasma: efficient solid-phase extraction and liquid chromatography/APCI-triple quadrupole mass spectrometry detection

Propofol (2,6-diisopropyl phenol) is widely used for the induction and maintenance of anesthesia. Analyses of its pharmacokinetics require simple and sensitive methods for quantitation of propofol in human plasma. Previously reported HPLC and GC methods are limited by cumbersome extraction steps. We describe a novel method that combines sample preparation by solid-phase extraction (SPE) with hydrophilic-lipophilic balance cartridges and analysis with a sensitive LC-APCI-triple quadrupole mass spectrometry (MS/MS) method for better quantitation. The absolute recovery of the analyte was greater than 96%. The limit of quantification for propofol in plasma at a signal-to-noise ratio of 10 was 5 ng/ml. The precision of the assay yielded coefficients of variation ranging from 2.9 to 5.3% and an accuracies of 99-105%. Our method advances the quantitative analysis of propofol in human plasma by combining simple, rapid and efficient SPE with specific and sensitive quantitation by HPLC with APCI-MS/MS detection.     

Evidence for similarities and differences in the biosynthesis of fungal sterols

The sterol composition of two ascomycetous fungi, Saccharomyces cerevisiae and Gibberella fujikuroi, was examined by chromatographic (TLC, GLC, and HPLC) and spectral (MS and 1H-NMR) methods. Of notable importance was that both fungi produced cholesterol and a homologous series of long chain fatty alcohols (C22 to C30). In addition to ergosterol two novel sterols, ergosta-5,7, 9(11), 22-tetraenol and ergosterol endoperoxide, were isolated as minor compounds in growth-arrested cultures of yeast and in mycelia of G. fujikuroi. 24-Ethylidenelanosterol was also detected in mycelia of G. fujikuroi. A shift in sterol biosynthesis was observed by treatment with 24 (RS), 25-epiminolanosterol (an inhibitor of the S-adenosylmethionine C-24 transferase) and by monitoring the sterol composition at various stages of development. The results are interpreted to imply that the genes for 24-desalkyl, e.g., cholesterol, and 24-alkyl sterols, e.g., 24 beta- methyl cholesterol and 24-ethyl cholesterol, are distributed (but not always expressed) generally throughout the fungi but the occurrence of one or another compounds is influenced by the fitness (structure and amount) for specific sterols to act functionally during fungal ontogeny; sterol fitness is coordinated with Darwinian selection pressures.     

Utilization of Δ5,7 - and Δ8-sterols by larvae of Heliothis zea

Larvae from two populations of Heliothis zea were reared on artificial diets containing various sterols, which supported suboptimal growth, and their tissue sterols were characterized in order to determine how these dietary sterols are utilized by this insect. The sterols studied included Δ^5,7-sterols (7-dehydrocholesterol or ergosterol), Δ⁸-sterols (lanosterol and/or 24-dihydrolanosterol), and a Δ⁵-sterol (4,4-dimethylcholesterol). Although larvae did not develop on 4,4-dimethylcholesterol, those fed primarily Δ⁸-4,4,14-trimethylsterols developed to the third instar. When the latter sterols were spared with cholesterol, the larvae reached the sixth instar and contained 4,4,14-trimethylsterols as well as cholesterol in their tissues. When larvae were fed 7-dehydrocholesterol, <1% of the larvae from one population developed to the sixth instar and these larvae contained 7-dehydrocholesterol as their principal sterol. The other larvae successfully completed their larval stage when they were transferred from the diet containing 7-dehydrocholesterol (or no sterol) to a diet containing cholesterol within at least 9 days. The sterol composition of larvae transferred from a diet containing cholesterol to a diet containing 7-dehydrocholesterol, after they had reached 60% of their final weight, was 54% cholesterol and 46% 7-dehydrocholesterol. The major sterol isolated from the tissues of the larvae fed ergosterol was also 7-dehydrocholesterol. Therefore, although the larva of H. zea can dealkylate and saturate the side chain of the Δ^5,7,22-24β-methylsterol, it carries out little metabolism of the B ring of the nucleus. These studies demonstrate that, when Δ^5,7- or Δ⁸-sterols are the principal sterols in the diet of H. zea, they are absorbed and incorporated into its tissues, although they slow the rate of growth and may prevent complete development of the larva.     

The effect of dietary cholesterol on the development of Hylemya brassicae

The influence of different concentrations of cholesterol upon the larval and postlarval development of Hylemya brassicae has been investigated using an artificial diet and axenic culture conditions.In contrast with other insects studied, H. brassicae larvae are extremely sensitive to cholesterol, even in minute concentrations, until after their second ecdysis. Concentrations of 0.1 to 0.$\text{4}$g/100 ml disturb the second moulting process in a typical way that could indicate an endocrine unbalance and result in a very high mortality during this process. When cholesterol at a concentration of 0.4g/100 ml is administered to the larvae after the second ecdysis, there is no longer an adverse effect on the further larval and postlarval development.The unusual sensitivity of young cabbage root fly larvae for cholesterol seems to point out some significant differences in the sterol requirements and metabolism of the species in comparison with other insects studied.     

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.     

Sterol/steroid metabolism and absorption in a generalist and specialist caterpillar: Effects of dietary sterol/steroid structure,mixture and ratio

Insects cannot synthesize sterols de novo, so they typically require a dietary source. Cholesterol is the dominant sterol in most insects, but because plants contain only small amounts of cholesterol, plant-feeding insects generate most of their cholesterol by metabolizing plant sterols. Plants almost always contain mixtures of different sterols, but some are not readily metabolized to cholesterol. Here we explore, in two separate experiments, how dietary phytosterols and phytosteroids, in different mixtures, ratios, and amounts, affect insect herbivore sterol/steroid metabolism and absorption; we use two caterpillars species – one a generalist (Heliothis virescens), the other a specialist (Manduca sexta). In our first experiment caterpillars were reared on two tobacco lines – one expressing a typical phystosterol profile, the other expressing high amounts/ratios of stanols and 3-ketosteroids. Caterpillars reared on the control tobacco contained mostly cholesterol, but those reared on the modified tobacco had reduced amounts of cholesterol, and lower total sterol/steroid body profiles. In our second experiment, caterpillars were reared on artificial diets containing known amounts of cholesterol, stigmasterol, cholestanol and/or cholestanone, either singly or in various combinations and ratios. Cholesterol and stigmasterol-reared moths were mostly cholesterol, while cholestanol-reared moths were mostly cholestanol. Moth tissue cholesterol concentration tended to decrease as the ratio of dietary cholestanol and/or cholestanone increased. In both moths cholestanone was metabolized into cholestanol and epicholestanol. Interestingly, M. sexta generated much more cholestanol than epicholestanol, while H. virescens did the opposite. Finally, total tissue steroid levels were significantly reduced in moths reared on diets containing very high levels of cholestanol. We discuss how dietary sterol/steroid structural differences are important with respect to sterol/steroid metabolism and uptake, including species-specific differences.     

Atmospheric pressure chemical ionization-mass spectrometry method to improve the determination of dansylated polyamines

Determination of polyamine pools is still a step impossible to circumvent in studies aimed at determining the pathophysiological role of natural polyamines. In addition, polyamine measurement in biological fluids and tissues may have clinical relevance, especially in cancer patients. Among the wide panel of analytical methods developed for the quantification of polyamines, high-performance liquid chromatographic (HPLC) separation of polyamines after derivatization with dansyl chloride remains the most commonly used method. In this work, we show that atmospheric pressure chemical ionization-mass spectrometry (MS) can be used to detect and quantify biologically relevant polyamines after dansylation, without chromatographic separation. Positive-ion mass spectra for each dansylated polyamine were generated after optimization by flow injection analysis (FIA). FIA coupled with MS detection by selected ion monitoring greatly increased the sensitivity of the polyamine detection. The method is linear over a wide range of polyamine concentrations and allows detection of quantities as low as 5 fmol. The FIA/MS method is about 50-fold more sensitive than the conventional HPLC/fluorimetry procedure. A good correlation (r>0.98) between these two methods was observed. The FIA/MS method notably reduces the time of analysis per sample to 1.5 min and turns out to be rapid, efficient, cost saving, reproducible, and sufficiently simple to allow its routine application.     

Incorporation of microalgae sterols by scallop Pecten maximus (L.) larvae

Changes in sterol composition of Pecten maximus larvae during the larval development stage with standard algal mixtures and unialgal diets were analysed. The sterol composition of four microalgae currently used in mollusc hatchery were also examined. Under standard algal conditions, the larvae quickly use the steryl ester from larvae reserves during the endotrophic and the mixotrophe phases. The preferential incorporation of Pavlova lutheri and T-Isochrysis sterols, rather than Skeletonema costatum sterols, during the larval development stage would indicate that S. costatum cells were poorly ingested and digested by larvae. Among the ingested sterols, cholesterol and stigmasterol were preferentially incorporated by the larvae. Conversely, the larvae appeared able to limit the incorporation of methylpavlovol, ethylpavlovol, and 4alpha-methylporiferasterol. In the unialgal experiment, the best growths were obtained with the diet richest in cholesterol (Chaetoceros calcitrans) and the best compromise of good growth and settlement rate was observed with the diet richest in C24 ethyl sterol. The selective incorporation of the cholesterol was confirmed by the larval rearing with C. calcitrans. The strong sterol dietary imprint in larvae corroborated the absence of an important capacity in P. maximus larvae to convert or biosynthesise sterol.