Marine 4-methyl sterols: synthesis of C-24 epimers of 4 alpha, 24-dimethyl-5 alpha-cholestan-3 beta-ol and 360 MHz 1HNMR comparisons to the natural product from Plexaura homomalla

Comparison of the highfield 1HNMR spectrum of 4 alpha, 24-dimethyl-5 alpha-cholestan-3 beta-o1 isolated by open column adsorptive chromatography and reversed-phase HPLC from P. homomalla with those of the corresponding synthetic 24 alpha and 24 beta compounds demonstrate that the gorgonian natural product is purely 24 beta, the same C-24 configuration found in sterols related to dinosterol and gorgosterol. 360 MHz 1HNMR data are also reported for synthetic 4 alpha, 24 beta-dimethyl-5 alpha-cholest-22E-en-3 beta-o1 (another P. homomalla natural product). The use of 1HNMR correlations in assigning C-24 configurations of 24-methyl marine sterols possessing various nuclei is examined and discussed. Analyses of the methyl sterol components of P. homomalla are tabulated and discussed with regard to origin and plausible biosynthetic interrelationships in light of the C-24 configurational findings.     

Gas chromatographic differentiation of 4-desmethyl-, 4-monomethyl- and 4,4-dimethyl-sterols. Part III: differentiation of sterols based on deltaROxo-values on four stationary phases.

The deltaROxo-values, i.e., ratios of retention data for 3-oxo derivatives to those for the corresponding free sterols have been determined for fourteen 4-desmethylsterols, six 4-monomethylsterols and twenty-one 4,4-dimethylsterols on SE-30, OV-17, QF-1 and HiEFF-8B stationary phases. The QF-1 phase is found superior to the others in affording the values which are the most effective for differentiation between the three sterol groups. Within the same sterol group, the values for individual sterol members on any of the stationary phases differ to some extent due to minor variations in their skeletal structure. 4-Desmethyl-delta-5-sterols have strikingly large deltaROxo-values and are distinguishable definitely from other members of the 4-desmethylsterol group. The relative deltaROxo-values, i.e., ratios of deltaROxo-values of individual sterols to the deltaROxo-values of beta-sitosterol, also have been calculated from the deltaROxo-values of individual sterols.     

The sterols of calcareous sponges (Calcarea, Porifera)

Sponges are sessile suspension-feeding organisms whose internal phylogenetic relationships are still the subject of intense debate. Sterols may have the potential to be used as independent markers to test phylogenetic hypotheses. Twenty representative specimens of calcareous sponges (class Calcarea, phylum Porifera) with a broad coverage within both subclasses Calcinea and Calcaronea were analysed for their sterol content. Two major pseudohomologous series were found, accompanied by some additional sterols. The first series encompassing conventional C(27) to C(29)Delta(5,7,22) sterols represented the major sterols, with ergosterol (ergosta-5,7,22-trien-3beta-ol, C(28)Delta(5,7,22)) being most prominent in many species. The second series consisted of unusual C(27) to C(29)Delta(5,7,9(11),22) sterols. Cholesterol occurred sporadically, mostly in trace amounts. The sterol patterns did not resolve intraclass phylogenetic relationships, namely the distinction between the subclasses, Calcinea and Calcaronea. This pointed towards major calcarean lipid traits being established prior to the separation of subclasses. Furthermore, calcarean sterol patterns clearly differ from those found in Hexactinellida, whereas partial overlap occurred with some Demospongiae. Hence, sterols only partly reflect the phylogenetic separation of Calcarea from both of the other poriferan classes that was proposed by recent molecular work and fatty acid analyses.     

Gas chromatographic differentiation of 4-desmethyl-, 4-monomethyl- and 4,4-dimethyl-sterols. Part II : differentiation of sterols based on deltaRAc-values on four stationary phases.

The deltaRAc-values, i.e., ratios of retention data for sterol acetates to those for the corresponding free sterols, were determined for fourteen 4-desmethylsterols, seven 4-monomethylsterols and twenty-two 4,4-dimethylsterols on SE-30, OV-17, QF-1 and HiEFF-8B stationary phases. The QF-1 phase is found sperior to the others in affording the values which are the most effective for differentiation between the three sterol groups. Within the same sterol group, the values for individual sterol members on any of the stationary phases differ to some extent due to minor variations in their skeletal structure. These differences are useful for distinguishing individual members of the same sterol group differing in their skeletal structure, particularly in the position of double bond. The relative deltaRAc-values, i.e., ratios of deltaRAc-values of individual sterols to the deltaRAc-value of a standard sterol, are found almost independent upon the operating conditions of gas liquid chromatography and may be regarded as a characteristic value of sterols on one and the same stationary phase.     

Seasonal and geographic variations of the sterols from the starfish asterias vulgaris (verrill)

The levels of the major sterols of the starfish $\text{Asterias vulgaris}$ collected at one location in Nova Scotia varied considerably from month to month. After spawning in June the levels of the sterols in the starfish were very low, but a rapid assimilation of dietary sterols allowed the total sterol level to increase approximately two-hundred-fold to the annual maximum in July. The levels of a few minor sterols were unaffected by the spawning process, and during this period they emerged as the major components of the sterol mixture. The sterol mixtures from samples collected at different locations were compared.     

Predicting the effect of steroids on membrane biophysical properties based on the molecular structure

The relationship between sterol structure and the resulting effects on membrane physical properties is still unclear, owing to the conflicting results found in the current literature. This study presents a multivariate analysis describing the physical properties of 83 steroid membranes. This first structure-activity analysis supports the generally accepted physical effects of sterols in lipid bilayers. The sterol chemical substituents and the sterol/phospholipid membrane physical properties were encoded by defining binary variables for the presence/absence of those chemical substituents in the polycyclic ring system and physical parameters obtained from phospholipid mixtures containing those sterols. Utilizing Principal Coordinates Analysis, the steroid population was grouped into five well-defined clusters according to their chemical structures. An examination of the membrane activity of each sterol structural cluster revealed that a hydroxyl group at C3 and an 8-10 carbon isoalkyl side-chain at C17 are mainly present in membrane active sterols having rigidifying, molecular ordering/condensing effects and/or a raft promoting ability. In contrast, sterol chemical structures containing a keto group at C3, a C4-C5-double bond, and polar groups or a short alkyl side-chain at C17 (3 to 7 atoms) are mostly found in sterols having opposite effects. Using combined multivariate approaches, it was concluded that the most important structural determinants influencing the physical properties of sterol-containing mixtures were the presence of an 8-10 carbon C17 isoalkyl side-chain, followed by a hydroxyl group at C3 and a C5-C6 double bond. Finally, a simple Logistic Regression model predicting the dependence of membrane activity on sterol chemical structure is proposed.     

Minor and trace sterols in marine invertebrates : VI. Occurrence and possible origins of sterols possessing unusually short hydrocarbon side chains

Sterols with biosynthetically unusually short side chains (fewer than eight carbon atoms expected for primary squalene cyclization products) have been identified in the extracts of numerous marine invertebrates. The structures of the short side chain and conventional side chain sterols have been determined for various species of Porifera and Coelenterata. Sterol structures were determined by comparison of their mass spectra and gas chromatographic retention times with those of authentic or synthetic samples. Evidence is presented supporting the natural occurrence of these compounds in the tissues of the marine invertebrates as opposed to formation by degradative processes during sample handling or laboratory work-up. The short side chain sterols were found to possess predominantly the androst-5-en-3β-ol nucleus with C-17 alkyl side chains ranging from zero to six carbon atoms. Concentrations of short side chain sterols range from trace levels to over 5% of the sterol mixture in various species. The possible origins of these short side chain sterols are evaluated in the light of current knowledge of sterol function, biosynthesis, dealkylation, microbial degradation, and autoxidation. Known sterol autoxidations are reviewed, and possible singlet oxygen and free radical mechanisms of sterol side chain autoxidation (at physiological temperatures) which may lead to sterols with shortened hydrocarbon side chain are suggested. The possible autoxidative generation of short side chain sterols from known marine sterols by the suggested mechanisms is evaluated through application of the REACT computer program. Predicted short side chains are tabulated for each parent marine sterol side chain and then compared with the compositions of the actual sterols found in the marine extracts examined. The possible natural environmental or in vivo autoxidative formation of the short side chain marine sterols is supported by these evaluations.     

A SURVEY OF THE STEROL COMPOSITION OF THE MARINE DINOFLAGELLATES KARENIA BREVIS,KARENIA MIKIMOTOI,AND KARLODINIUM MICRUM: DISTRIBUTION OF STEROLS WITHIN OTHER MEMBERS OF THE CLASS DINOPHYCEAE1

The sterol composition of different marine microalgae has been examined to determine the utility of sterols as biomarkers to distinguish members of various algal classes. For example, members of the class Dinophyceae possess certain 4‐methyl sterols, such as dinosterol, which are rarely found in other classes of algae. The ability to use sterol biomarkers to distinguish certain dinoflagellates such as the toxic species Karenia brevis Hansen and Moestrup, responsible for red tide events in the Gulf of Mexico, from other species within the same class would be of considerable scientific and economic value. Karenia brevis has been shown by others to possess two major sterols, (24S)‐4α‐methyl‐5α‐ergosta‐8(14),22‐dien‐3β‐ol (ED) and its 27‐nor derivative (NED), having novel structures not previously known to be present in other dinoflagellates. This prompted the present study of the sterol signatures of more than 40 dinoflagellates. In this survey, sterols with the properties of ED and NED were found in cultures of K. brevis and shown also to be the principal sterols of Karenia mikimotoi Hansen and Moestrup and Karlodinium micrum Larsen, two dinoflagellates closely related to K. brevis. They are also found as minor components of the more complex sterol profiles of other members of the Gymnodinium/Peridinium/Prorocentrum (GPP) taxonomic group. The distribution of these sterols is consistent with the known close relationship between K. brevis, K. mikimotoi, and K. micrum and serves to limit the use of these sterols as lipid biomarkers to a few related species of dinoflagellates.     

Lipid acyl chain-dependent effects of sterols in Acholeplasma laidlawii membranes.

Acholeplasma laidlawii was grown with different fatty acids for membrane lipid synthesis (saturated straight- and branched-chain acids and mono- and di-unsaturated acids). The ability of 12 different sterols to affect cell growth, lipid head group composition, the order parameter of the acyl chains, and the phase equilibria of in vivo lipid mixtures was studied. The following two effects were observed with respect to cell growth: with a given acyl chain composition of the membrane lipids, growth was stimulated, unaffected, reduced, or completely inhibited (lysis), depending on the sterol structure; and the effect of a certain sterol depended on the acyl chain composition (most striking for epicoprostanol, cholest-4-en-3-one, and cholest-5-en-3-one, which stimulated growth with saturated acyl chains but caused lysis with unsaturated chains). The three lytic sterols were the only sterols that caused a marked decrease in the ratio between the major lipids monoglucosyldiglyceride and diglucosyldiglyceride and hence a decrease in bilayer stability when the membranes were enriched in saturated (palmitoyl) chains. With these chains correlations were found for several sterols between the glucolipid ratio and the order parameter of the acyl chains, as well as the lamellar-reversed hexagonal phase transition, in model systems. A shaft experiment revealed a marked decrease in the ratio of monoglucosyldiglyceride to diglucosyldiglyceride with the lytic sterols in unsaturated (oleoyl) membranes. The two cholestenes induced nonlamellar phases in in vivo mixtures of oleoyl A. laidlawii lipids. The order parameters of the oleoyl chains were almost unaffected by the sterols. Generally, the observed effects cannot be explained by an influence of the sterols on the gel-to-liquid crystalline phase transition.     

Ternary gradient elution markedly improves silver-ion high performance liquid chromatography of unsaturated sterols

A wide variety of unsaturated sterols can accumulate in eukaryotic cells as a consequence of normal metabolism, genetic disorders, and actions of enzyme inhibitors. Resolving these sterol mixtures into individual components by conventional chromatographic methods is inefficient because unsaturated sterols differ little in polarity, hydrophobicity, and volatility. Although sterol mixtures are well-resolved by silver-ion high performance liquid chromatography (Ag(+)-HPLC), existing methods require derivatization to acetates for best results, and the isocratic mobile phases lead to long analysis times and low sensitivity for late-eluting sterols. We show that these problems can be overcome with ternary gradient elution using acetone, hexanes, and acetonitrile. Separation of a mixture of 20 underivatized sterols gave dramatically shortened analysis times, with good peak shapes for both early- and late-eluting components. In a similar separation of blood sterols from a patient with Smith-Lemli-Opitz syndrome, the band for 7-dehydrocholesterol was much narrower than with isocratic elution. Column re-equilibration was rapid, and the separations could be monitored with ultraviolet spectroscopy at 210 nm, which affords universal, non-destructive detection of unsaturated sterols. Also discussed are retention mechanisms and reproducibility of Ag(+)-HPLC separations. The overall results represent a major advance in chromatographic methods for resolving mixtures of unsaturated sterols differing in the number and position of olefinic bonds.     

Cholesterol and ergosterol superlattices in three-component liquid crystalline lipid bilayers as revealed by dehydroergosterol fluorescence.

We have examined the fractional sterol concentration dependence of dehydroergosterol (DHE) fluorescence in DHE/cholesterol/dimyristoyl-L-alpha-phosphatidylcholine (DMPC), DHE/ergosterol/DMPC and DHE/cholesterol/dipalmitoyl-L-alpha-phosphatidylcholine (DPPC) liquid-crystalline bilayers. Fluorescence intensity and lifetime exhibit local minima (dips) whenever the total sterol mole fraction, irrespective of the DHE content, is near the critical mole fractions predicted for sterols being regularly distributed in hexagonal superlattices. This result provides evidence that all three of these naturally occurring sterols (e.g., cholesterol, ergosterol, and DHE) can be regularly distributed in the membrane and that the bulky tetracyclic ring of the sterols is the cause of regular distribution. Moreover, at the critical sterol mole fractions, the steady-state anisotropy of DHE fluorescence and the calculated rotational relaxation times exhibit distinct peaks, suggesting that membrane free volume reaches a local minimum at critical sterol mole fractions. This, combined with the well-known sterol condensing effect on lipid acyl chains, provides a new understanding of how variations in membrane sterol content change membrane free volume. In addition to the fluorescence dips/peaks corresponding to hexagonal superlattices, we have observed intermediate fluorescence dips/peaks at concentrations predicted by the centered rectangular superlattice model. However, the 22.2 mol% dip for centered rectangular superlattices in DHE/ergosterol/DMPC mixtures becomes diminished after long incubation (4 weeks), whereas on the same time frame the 22.2 mol% dip in DHE/cholesterol/DMPC mixtures remains discernible, suggesting that although all three of these sterols can be regularly distributed, subtle differences in sterol structure cause changes in lateral sterol organization in the membrane.     

Free sterols from the holothurians Synapta maculata, Cladolabes bifurcatus and Cucumaria sp

Free sterol fractions from the holothurians (sea cucumbers) Synapta maculata, Cladolabes bifurcatus and Cucumaria sp. have been isolated and studied by HPLC, GLC, GLC-MS and NMR methods. Forty seven sterols were identified, including several rare ones. In contrast with previously studied holothurians, the presence of a minor amount of Delta7 sterols was indicated in the sterols of S. maculata. This animal contains predominantly Delta(9(11))sterols as well as an abnormally high concentration of Delta5 sterols. In C. bifurcatus and Cucumaria sp., 14alpha-methyl and 4alpha,14alpha-dimethyl-Delta(9(11))sterols were found to be the main sterol constituents. Peculiarities of sterol distribution and the relationship between sterol compositions and taxonomic positions, ecology and toxicity of the corresponding sea cucumbers were discussed.     

Minor and trace sterols in marine invertebrates XVI. 3 epsilon-hydroxymethyl-A-nor-5 alpha-gorgostane, a novel sponge sterol

The free sterol mixture of the sponge Stylotella agminata contained a series of 3-hydroxymethyl-A-nor sterols of 5 alpha-cholestane, 5 alpha-cholest-22-ene, 24-methyl-5 alpha-cholestane, 24-ethyl-5 alpha-cholestane, and 24-ethyl-5 alpha-cholest-22-ene. A new cyclopropane-containing sterol was isolated and shown to be 3 epsilon-hydroxymethyl-A-nor-5 alpha-gorgostane. Sterols with conventional nuclei were also present as minor constituents;     

Sterols of the ‘dinotom’ Durinskia baltica (Dinophyceae) are of dinoflagellate origin

‘Dinotoms’ are a relatively small group of dinoflagellates with aberrant tertiary plastids of diatom origin, thus differing from the majority of photosynthetic dinoflagellates which possess the carotenoid pigment peridinin and have secondary plastids of red algal origin. As part of our laboratory's continuing efforts to examine such unusual dinoflagellates in the search for clues to the evolution of their lipid compositions, we have examined the sterol composition of the dinotom Durinskia baltica. As such, we here compared its sterols to those of the previously examined dinotom, Kryptoperidinium foliaceum, more broadly to other photosynthetic, peridinin-containing dinoflagellates, and to the diatom genus Nitzschia, which is the presumed ancestor of the D. baltica dinotom plastid. Sterols are ringed lipids, common to eukaryotes, thought to reinforce phospholipid bilayers. Many peridinin-containing dinoflagellates have sterol compositions which are enriched by the presence of cholesterol (cholest-5-en-3β-ol) and 4α-methyl-substituted sterols such as dinosterol (4α,23,24-trimethyl-5α-cholest-22E-en-3β-ol); this has also been found to be true for K. foliaceum despite its aberrant plastid ancestry. Our objective was to determine if this is also true for D. baltica as only the second dinotom to have its sterols characterized in detail, and to determine if there is any indication of prominent sterols which are uncommon to dinoflagellates, possibly originating from the diatom endosymbiont, as has been demonstrated previously with K. foliaceum and D. baltica chloroplast-associated galactolipids of clear diatom origin. Our results demonstrate that like K. foliaceum, the major sterols of D. baltica are cholesterol, dinosterol, and other 4α-methyl-substituted sterols common to dinoflagellates. Although there were a number of minor sterols, none were found with obvious origin from the diatom endosymbiont, indicating that most originated with the dinoflagellate host itself, most likely before acquisition of the diatom tertiary plastid.     

Sterols of Amphidinium species in the Operculatum Clade: Predominance of cholesterol instead of Δ8(14) sterols previously considered Amphidinium-specific biomarkers

The dinoflagellates Amphidinium carterae and Amphidinium corpulentum have been previously characterized as having Δ⁸⁽¹⁴⁾-nuclear unsaturated 4α-methyl-5α-cholest-8(14)-en-3β-ol (C_28:1) and 4α-methyl-5α-ergosta-8(14),24(28)-dien-3β-ol (amphisterol; C_29:2) as predominant sterols, where they comprise approximately 80% of the total sterol composition. These two sterols have hence been considered as possible major sterol biomarkers for the genus. Here, we have examined the sterols of four recently identified species of Amphidinium (Amphidinium fijiense, Amphidinium magnum, Amphidinium theodori, and Amphidinium tomasii) that are closely related to Amphidinium operculatum as part of what is termed the Operculatum Clade to show that each species has its sterol composition dominated by the common dinoflagellate sterol cholesterol (cholest-5-en-3β-ol; C_27:1), which is found in many other dinoflagellate genera, rather than Δ⁸⁽¹⁴⁾ sterols. While the Δ⁸⁽¹⁴⁾ sterols 4α-methyl-5α-cholest-8(14)-en-3β-ol and 4α,23,24-trimethyl-5α-cholest-8(14),22E-dien-3β-ol (C_30:2) were present as minor sterols along with another common dinoflagellate sterol, 4α,23,24-trimethyl-5α-cholest-22E-en-3β-ol (dinosterol; C_30:1), in some of these four species, amphisterol was not conclusively observed. From a chemotaxonomic perspective, while this does reinforce the genus Amphidinium's ability to produce Δ⁸⁽¹⁴⁾ sterols, albeit here as minor sterols, these results demonstrate that caution should be used when considering Δ⁸⁽¹⁴⁾ sterols, especially amphisterol, as Amphidinium-specific biomarkers within these species where cholesterol is the predominant sterol.     

Marine sterols. XV. Sterols of some oceanic holothurians

Mature and immature individuals of the deep-water detritus-feeder Benthodytes lingua Perrier and mature specimens of the mid-water detritus-feeders Stichopus tremulus (Gunnerus) and Mesothuria verrilli (Theel) have been analysed by gas chromatographic-mass spectrometric techniques for their component sterols. The results indicate a highly variable sterol and stanol composition amongst holothurians irrespective of water depth, probable diet or even life stage. It is suggested that the complex sterol mixtures found reflect specific structural requirements of holothurians for both component sterols and stanols.     

Influence of the nature of the sterol on the behavior of palmitic acid/sterol mixtures and their derived liposomes

The phase behavior of mixtures formed with palmitic acid (PA) and one of the following sterols (dihydrocholesterol, ergosterol, 7-dehydrocholesterol, stigmasterol and stigmastanol), in a PA/sterol molar ratio of 3/7, has been characterized by IR and ²H NMR spectroscopy at different pH. Our study shows that it is possible to form liquid-ordered (lo) lamellar phases with these binary non-phospholipid mixtures. The characterization of alkyl chain dynamics of PA in these systems revealed the large ordering effect of the sterols. It was possible to extrude these systems, using standard extrusion techniques, to form large unilamellar vesicles (LUVs), except in the case of ergosterol-containing mixture. The resulting LUVs displayed a very limited passive permeability consistent with the high sterol concentration. In addition, the stability of these PA/sterol self-assembled bilayers was also found to be pH-sensitive, therefore, potentially useful as nanovectors. By examining different sterols, we could establish some correlations between the structure of these bilayers and their permeability properties. The structure of the side chain at C17 of the sterol appears to play a prime role in the mixing properties with fatty acid.     

Regulation of partitioned sterol biosynthesis in Saccharomyces cerevisiae.

Using yeast strains with null mutations in structural genes which encode delta-aminolevulinic acid synthetase (HEM1), isozymes of 3-hydroxy-3-methylglutaryl coenzyme A (HMG1 and HMG2), squalene epoxidase (ERG1), and fatty acid delta 9-desaturase (OLE1), we were able to determine the effect of hemes, sterols, and unsaturated fatty acids on both sterol production and the specific activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) in Saccharomyces cerevisiae. We found that the HMGR isozymes direct essentially equal amounts of carbon to the biosynthesis of sterols under heme-competent conditions, despite a huge disparity (57-fold) in the specific activities of the reductases. Our results demonstrate that palmitoleic acid (16:1) acts as a rate-limiting positive regulator and that ergosterol acts as a potent inhibitor of sterol production in strains which possess only the HMGR1 isozyme (HMG1 hmg2). In strains which contain only the HMGR2 isozyme (hmg1 HMG2), sterol production was inhibited by oleic acid (18:1) and to a lesser degree by ergosterol. The specific activities of the two reductases (HMGR1 and HMGR2) were found to be differentially regulated by hemes but not by ergosterol, palmitoleic acid, or oleic acid. The disparate effects of unsaturated fatty acids and sterols on these strains lead us to consider the possibility of separate, compartmentalized isoprenoid pathways in S. cerevisiae.     

Sterol metabolic constraints as a factor contributing to the maintenance of diet mixing in grasshoppers (Orthoptera: Acrididae)

Sterols are essential nutrients for all arthropods, including grasshoppers, but metabolic constraints may limit which sterols can support normal growth and development. In the firsts part of this study, a comparative experiment, which included five different species of grasshoppers (Orthoptera: Acrididae) representing three separate taxonomic groups, was performed to determine how widespread sterol metabolic constraints are within the Acrididae. Grasshoppers were reared on artificial diets containing sterols that differed in the position of double bonds within the sterol structure, and various life history traits were measured. Sterols with double bonds at position 7, within the sterol nucleus, and/or at position 22, on the cholestane side chain, failed to support development to the adult stage for any of the five species. In addition, grasshoppers reared on sterols with these configurations often had extended developmental times and reduced growth rates in the first and second stadium compared with grasshoppers reared on sitosterol or cholesterol diets. In the second half of this study, we examined how mixtures of suitable and unsuitable sterols influenced survival, growth, and development. Artificial foods containing mixtures of suitable and unsuitable sterols were fed to the highly polyphagous grasshopper Schistocerca americana. Results suggest that survival and performance of this grasshopper suffer as the concentration of unsuitable sterols increases and as the ratio of suitable to unsuitable sterols in the diet decreases. We review the literature to document variation in plant sterol profiles and propose that constraints on sterol metabolism may contribute to the maintenance of diet mixing in the Acrididae.     

Sterols in Germinating Seeds and Developing seedlings of Longleaf Pine, Pinus palustris

Sterols in germinating embryos and young seedlings of longleaf pine (Pinus palustris Mill.) were identified and quantities determined for different periods after germination. Sterol analyses were performed by gas-liquid chromatography (GLC) and verified by combination of GLC-mass spectrometry. Campesterol and β-sitosterol were two major sterols which accounted for most of the sterol composition while stigmasterol was present in very small amounts. No cholesterol was revealed by GLC-mass spectrometry although there was a minor peak appearing on the sterol gas-liquid chromatograms with a retention time close to that of authentic cholesterol. By fractionation, three different forms of sterols were obtained: steryl esters, steryl glycosides, and free sterols. The sterols were mainly found in the esterified fraction, while steryl glycosides and free sterols only made up a small portion of the total sterol value. The total sterol content in general increased during seedling development, and this increase reflected mainly a change in steryl esters. The low levels of both free and glycosidic sterols remained nearly unchanged throughout the experimental germination period.