L. John Goad

The 220 MHz NMR spectra of phytosterols

The 220MHz NMR spectra of forty two steroids are reported. Eight pairs of C-24 epimers (24α- and 24β) and two pairs of double bond isomers (cis and trans) can be distinguished by this technique. The influence of substituents, solvents and stereochemistry on methyl group chemical shifts is discussed.

Analysis of sterols

1. Nomenclature and Biosynthesis of Sterols and Related Compounds. 2. Extraction of Sterols from Tissues. 3. Initial Separation Methods. 4. HPLC of Sterols. 5. Gas-Liquid Chromatography of Sterols. 6. Infra- Red and Ultra-Violet Spectroscopy of Sterols. 7. Mass Spectrometry of Sterols. 8. 1H NMR Spectroscopy of Sterols. 9. 13C NMR Spectroscopy of Sterols. 10. One-Dimensional and Two-Dimensional NMR Spectroscopy of Sterols. 11. X-Ray Crystallography of Sterols. 12. Sources of Sterols. Tables of Physical Data. References. Index.

Analysis of organic residues of archaeological origin by high-temperature gas chromatography and gas chromatography-mass spectrometry

Organic residues are extracted from materials of archaeological interest by solvent extraction and subjected directly to high-temperature gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS). The use of high-temperature GC allows intact acyl lipids, e.g., triacylglycerols, diacylglycerols, monoacylglycerols and wax esters, to be analysed without prior degradation (e.g., saponification) to release constituent fatty acids and alcohols. Trimethylsilylation is employed to block protic sites in free fatty acids and hydroxylated components. The data obtained from temperature programmed GC and GC-MS analyses, employing immobilised apolar (dimethyl polysiloxane type) stationary phases, provide essential compositional information that would be lost if the more conventional degradative approach to acyl lipid analysis was adopted.

EFFECTS OF ANTIMYCOTIC AZOLES ON GROWTH AND STEROL BIOSYNTHESIS OF LEISHMANIA PROMASTIGOTES

Promastigotes of 36 World Health Organization reference (and other) strains of 6 species and 10 subspecies of Leishmania were cultured in the presence of 3 antimycotic azole drugs (ketoconazole, itraconazole, fluconazole) and their population growth determined. A representative of each subspecies was also analyzed for its sterol composition. For all strains the order of azole drug activity with respect to both growth and sterol biosynthesis inhibition was itraconazole greater than or equal to ketoconazole greater than fluconazole. The inhibitory actions of the three azole drugs were greater on L. donovani and L. braziliensis subspecies and on L. mexicana amazonensis than on L. aethiopica, L. major, L. tropica and L. mexicana mexicana. The nature of the changes in sterol composition caused by the drugs was the same for all strains. The normal, major endogenous sterols of the promastigotes (5-dehydroepisterol and ergosterol) were reduced in amount to 1-2% of the total free sterols and were replaced by endogenous 14 alpha-methyl sterols and exogenous cholesterol. The changes occurred rapidly, were drug concentration dependent and coincided with growth inhibition. Six strains of those Leishmania species less sensitive to the azole drugs could be subcultured indefinitely at reduced growth rates in the presence of a ketoconazole concentration causing the same extraordinary alterations in sterol composition. This suggested that the bulk membrane functions of sterols in leishmanias can be served by 14 alpha-methyl sterols and cholesterol, albeit imperfectly, while traces of 14 alpha-desmethyl sterols are needed for uncharacterized metabolic functions.

Sterol requirements and paclobutrazol inhibition of a celery cell culture

Abstract The triazole plant growth regulator paclobutrazol was an effective inhibitor of growth of a celery cell suspension culture. Exposure of the celery cells to paclobutrazol caused inhibition of sterol biosynthesis at the 14α-demethylation step and there was an accumulation of several 14α-methylsterols [obtusifoliol, cycloeucalenol, cycloartenol, 14α,24-dimethylcholest-8-en-3β-ol and 14α,24-dimethylcholesta-8,24(28)-dien-3β-ol] with an accompanying decrease in the sitosterol and stigmasterol content of the cells. There was a marked fall in the stigmasterol: sitosterol ratio. Addition of paclobutrazol during the linear phase of growth (day 6) caused a rapid cessation of cell division. Obtusifoliol, added on day six to the growing celery cultures at low concentrations (0.05 to 5 μM), also stopped cell division but at the lowest concentration an increase in dry weight continued to day 10 before starting to decline. 14α-Methylcholest-9(11)-en-3β-ol was an effective inhibitor of cell growth but other 14α-methylsterols possessing a 4β-methyl group and/or a 9β,19-cyclopropane ring (lanosterol, dihydrolanosterol, cycloartenol, cycloeucalenol) had little or no effect on cell growth. The inhibition of celery cell growth by paclobutrazol could be partially reversed by addition of 50 μM cholesterol to the growth medium. Complete restoration of growth was achieved by addition of 50 μM stigmasterol or by 50 μM cholesterol plus a low concentration (0.05 μM to 5 μM) of stigmasterol. Stigmasterol alone at these low concentrations was ineffective in promoting growth of the paclobutrazol treated cells. These results reveal that the celery cells have two requirements for sterols. One is for relatively large amounts of sterol and may be associated with membrane elaboration. The other essential role, termed ‘trigger’, requires trace amounts of sterol with a 24α-ethyl side chain such as stigmasterol. Thus, plants have sterol requirements for cell proliferation which resemble those demonstrated for fungi. The significance of these results in relation to the mode of action of azole plant growth regulators is discussed.

Sterols of Leishmania species, implications for biosynthesis

The major sterol of promastigotes of stocks of Leishmania tropica, L. donovani and 3 subspecies of L. mexicana has been identified as ergosta-5,7,24(28)-trien-3 beta-ol; and of an L. major stock as ergosta-7,24(28)-dien-3 beta-ol. 24-Methylcholesta-5,7,22-trien-3 beta-ol and 24-ethylcholesta-5,7,22-trien-3 beta-ol were minor constituents, and traces of ergosta-5,7,22,24(28)-tetraen-3 beta-ol and a C27-diene were also recognized in some species. Lanosterol and 4,4-dimethylcholesta-8,24-dien-3 beta-ol were detected in all species studied, and squalene was identified in a stock of L. tropica. The sterol composition of members of the genus Leishmania and the sterol biosynthetic pathways it implies are characteristic of yeast and other fungi.

Occurrence of (24S)-24-methylcholesta-5, 22E-dien-3β-ol in the diatom Phaeodactylum tricornutum

The principal sterol of the marine diatom Phaedactylum tricornutum was identified as (24S)-24-methylcholesta-5,22E-dien-3β-ol. Two deuterium atoms were incorporated into this sterol when the diatom was cultured in the presence of [CD3]methionine indicating a 24-methylene intermediate.

Strategy for the analysis of steryl esters from plant and animal tissues

Methods are described for the analysis of intact steryl esters present in complex mixtures isolated from plant or animal tissues. A preliminary examination by analytical thin-layer chromatography (TLC) and capillary column gas chromatography-mass spectrometry (GC-MS) under electron impact (EI) ionisation reveals the complexity of the mixture and the nature of the steryl moieties. Preparative TLC is then utilised to separate the steryl esters into two broad groups, containing fatty acyl moieties of shorter (C2-C8) or longer chain length (C10-C22). The shorter-chain fatty acyl steryl esters are separated by adsorption high-performance liquid chromatography (HPLC) on a LiChrosorb Silica-60 column. The steryl esters with longer-chain fatty acyl moieties are analysed by reversed-phase HPLC on either an Ultrasphere ODS, 5-micron, or a S3 Spherisorb ODS, 3-micron, column. Steryl esters with unsaturated fatty acyl moieties are eluted with the shorter-chain fatty acyl steryl esters. The presence of the unsaturated fatty acyl esters can be monitored by analytical argentation TLC, which will also reveal the degree of unsaturation. The steryl esters are fractionated into the saturated, mono-, di-, tri- and polyene acyl types by preparative medium-pressure liquid chromatography on a column of 10% AgNO3-silica gel. Each of these steryl ester types can then be resubmitted to reversed-phase HPLC or analysed by GC-MS on a short fused-silica capillary column with a bonded phase of the OV-1 type. GC-MS on a magnetic-sector instrument under negative-ion chemical ionisation conditions with ammonia as the reagent gas produces fragment ions for both the steryl and fatty acyl moieties, thus permitting identification of the individual intact steryl esters. These various methods are illustrated by analyses of the steryl ester mixtures obtained from human plasma, barley seedlings, palm oil and rape seed oil.

Sterols of the siphonous marine alga Codium fragile

Abstract The marine siphonous green alga, Codium fragile , was shown to contain two 25-methylene sterols. These were identified as (24 S )-24-ethylcholesta-5.25-dien-3β-ol and the previously unknown (24 S )-24-methylcholesta-5,25-dien-3β-ol for which the trivial name codisterol is proposed.

Effects of ketoconazole on sterol biosynthesis by Leishmania mexicana mexicana amastigotes in murine macrophage tumor cells.

Murine macrophage tumor cells infected with Leishmania mexicana mexicana were exposed to the antimycotic drug ketoconazole and to [2-14C]mevalonate, then the amastigotes were isolated, collected, purified, and their free sterols were analyzed by chromatographic and mass spectrometric methods. Control amastigotes contained as products of de novo biosynthesis C28 4-desmethyl sterols (episterol, 5-dehydroepisterol), C29 4-desmethyl sterols (stigmasta-7,24 (28)-dien-3 beta-ol, stigmasta-5,7,24(28)-trien-3 beta-ol), 4-methyl sterols (4 alpha, 14 alpha-dimethylzymosterol, obtusifoliol) and a 4,4-dimethyl sterol (lanosterol). Present also were macrophage sterols (cholesterol, desmosterol) and a putative product of the C-24 alkylation of desmosterol by amastigotes (24-methylenecholesterol). Amastigotes from macrophages exposed to ketoconazole showed notable changes in the proportions, concentrations and specific activities of their free sterols; increased for 4 alpha, 14 alpha-dimethylzymosterol and decreased for the endogenous C28 and C29 4-desmethyl sterols. Such changes were observed at a ketoconazole concentration as low as 0.01 microgram ml-1. By contrast, uninfected macrophages accumulated only small amounts of lanosterol of high specific activity at a ketoconazole concentration of 10 micrograms ml-1. the ketoconazole-induced alterations in amastigote sterols parallel those previously reported in fungi and L. m. mexicana promastigotes, and suggest a biochemical mechanism for the anti-leishmanial activity of the drug in which changes in sterol composition are linked to disturbances of cell membrane structure and function, and hence to cytotoxicity.