William R. Lusby

Separation and quantitation of carotenoids in foods

Publisher Summary This chapter describes high-performance liquid chromatography (HPLC) procedures that allow effective separation and accurate quantitation of individual carotenoids in commonly consumed foods. Carotenoids are one of the most common dietary agents that have been studied as cancer preventive agents. Although the number of naturally occurring carotenoids isolated from various sources is in excess of 600, the number of carotenoids that are abundant in common foods is less than 50. HPLC has been shown to be the most efficient technique for the analysis of sensitive compounds in complex food extracts. Numerous HPLC conditions have been developed to separate the carotenoids in extracts of natural products. Such HPLC procedures can be highly advantageous and provide valuable information on the identity and the levels of these compounds in their natural state in foods. However, HPLC procedures that can be universally used for the separation and quantification of carotenoids in extracts of foods are lacking. The HPLC profiles of extracts from fruits and vegetables are illustrated in chapter.

Analysis of sterol esters by capillary gas chromatography-electron impact and chemical lonization-mass spectrometry

Synthetic mixtures of C40 to C47 sterol esters in groups of 7 esters were effectively separated and analyzed by capillary gas chromatography-mass spectrometry. Ammonia chemical ionization of all 20 sterol esters analyzed at a source block temperature of 120 C yielded (M+NH4)+ and (M+H-RCO2H)+ ions of high abundance or as base peak, thereby indirectly indicating the molecular weights of the ester and the sterol and acid moieties. Ammonia CI spectra of all esters containing a Δ5-sterol moiety exhibited in addition to the above 2 ions an M+NH4-RCO2 H fragment. At a source block temperature of 150 C, M+H-RCO2 H fragment was the base peak for all esters, and there was little or no indication of an (M+NH4)+ adduct ion. Protonated molecules were not observed for any esters analyzed by methane or isobutane CI. Molecular ions of 3–14% intensity were obtained for only 3 of the esters analyzed by electron impact; they contained a Δ7-bond in the sterol nucleus, and the acid moiety was either saturated normal or branched chain or contained a single double bond. The base peak was a function of both the acid and sterol moieties of the sterol ester. The esters containing both saturated straight chain acid and saturated sterol moieties exhibited a base peak at m/z 215. The Δ5-sterol esters with saturated branched or straight chain acid moieties exhibited base peaks at M-RCO2 H. Other ions also were of diagnostic value.

The glycosylceramides of the nematodeCaenorhabditis elegans contain an unusual, branched-chain sphingoid base

Caenorhabditis elegans was cultured in semi-defined medium containing yeast extract, soy peptone, glucose, hemoglobin, Tween 80, and sitosterol. Monoglycosylceramides were chromatographically purified from nematode extracts. Their structures were elucidated with mass spectrometry, nuclear magnetic resonance spectroscopy, and analysis of methanolysis products of the parent cerebrosides. The glycosylceramides were unusual in that the only long-chain sphingoid base detected was aniso-branched compound with a C-4 double bond (i.e., 15-methyl-2-aminohexadec-4-en-1,3-diol). Glucose was the only sugar moiety detected. The fatty acids consisted of a series of primarily straint-chain, saturated, 2-hydroxylated C20–C26 acids; someiso-branched analogs also occurred. The sphingomyelins ofC. elegans were also hydrolyzed, and the sameiso-branched C17 compound was the only sphingoid base detected. This is the first structural analysis of a nematode glycosphingolipid and the first report of an organism in which the long-chain sphingoid bases are entirelyiso-branched.

Separation and quantification of carotenoids in human plasma

Publisher Summary This chapter discusses separation and quantification of carotenoids in human plasma. A number of studies have reported an inverse relationship between the high consumption of certain fruits and vegetables and a lower incidence of several types of cancer. The first successful separation of carotenoids from an extract of human serum was performed by using nonaqueous reversed-phase chromatographic conditions on a Zorbax ODS column, with a mixture of acetonitrile, dichloromethane, and methanol as eluent. Under these conditions, six carotenoids were separated and identified in an extract from human serum: lutein, zeaxanthin, β cryptoxanthin, lycopene, α -carotene, and β -carotene. This chapter describes the methodologies for detailed separation and quantitation of 18 carotenoids along with vitamin A and 2 forms of vitamin E in human plasma by high-performance liquid chromatographic (HPLC) on reversed-phase and silica-based nitrilebonded columns.

Sterol metabolism in the nematodeCaenorhabditis elegans

The metabolism of various dietary sterols and the effects of an azasteroid on sitosterol metabolism in the free-living nematodeCaenorhabditis elegans was investigated. The major unesterified sterols ofC. elegans in media supplemented with sitosterol, cholesterol or desmosterol included 7-dehydrocholesterol (66.5%, 40.5%, 31.2%, respectively), cholesterol (6.7%, 52.3%, 26.9%), lathosterol (4.4%, 3.6%, 1.7%) and 4α-methylcholest-8(14)-en-3β-ol (4.2%, 2.1%, 3.8%). Esterified sterols, representing less than 20% of the total sterols, were somewhat similar except for a significantly higher relative content of 4α-methylcholest-8(14)-en-3β-ol (23.3%, 23.4%, 10.6%). ThusC. elegans not only removes the substituent at C24 of dietary sitosterol but possesses the unusual ability to produce significant quantities of 4α-methylsterols. WhenC. elegans was propagated in medium supplemented with sitosterol plus 5 μg/ml of 25-azacoprostane hydrochloride, the azasteroid strongly interfered with reproduction and motility ofC. elegans and strongly inhibited the Δ24-sterol reductase enzyme system; excluding sitosterol, the major free sterols of azacoprostane-treatedC. elegans were cholesta-5, 7, 24-trien-3β-ol (47.9%), desmosterol (9.4%), fucosterol (2.1%) and cholesta-7,24-dien-3β-ol (2.0%). These 4 sterols are likely intermediates in the metabolism of sitosterol inC. elegans.

STEROLS OF SOME DIATOMS

Abstract Sterols were identified from seven species of axenically cultured diatoms which may be used for oyster food. Five species contained 24-methylenecholesterol as the major sterol. Stigmasterol was the principal sterol in Amphora coffaeformis while 24-ethylcholesterol was the major sterol in Navicula pelliculosa . Each of the four species of the order Centrales had 24-methylcholesterol, which was all 22-dihydrobrassicasterol (24β-methyl epimer). The 24-methylcholesterol from Nitzschia brevirostris (order Pennales) was all campesterol (24α-methyl epimer). From this and previously published works on diatom sterols, it appears that sterols from the order Centrales are 24β-oriented and those from the order Pennales are 24α-oriented.

Effects of triarimol, tridermorph and triparanol on sterol biosynthesis in carrot, tobacco and soybean suspension cultures

The effects of triarimol, tridemorph and triparanol on sterol biosynthesis in carrot, tobacco and soybean suspension cultures were studied. The 3 plant species normally contain campesterol, stigmasterol and sitosterol as major sterols. Triarimol inhibited demethylation at C 14 and the second alkylation of the side chain in all 3 species. The primary effects of tridemorph were the inhibition of the opening of the 9β,19-cyclopropane ring and the second alkylation of the side chain. Triparanol treatments resulted in the accumulation of 14α-methyl sterols, and the inhibition of second alkylation in the side chain in carrot and tobacco cultures. Cyclopropyl sterols also accumulated in carrot and tobacco cultures treated with triparanol. Triparanol did not alter the sterol composition of soybean cultures except for decreasing concentrations of campesterol and stigmasterol and increasing amounts of sitosterol.

Novel nuclear methylation of sterols by the nematode caenorhabditis elegans

Caenorhabditis elegans possesses a unique sterol methylation pathway not reported to occur in any other organism and also removes the C-24 ethyl group of sitosterol (a plant sterol). This nematode produced substantial quantities of 4 alpha-methyl-5 alpha-cholest-8(14)-en-3 beta-ol and smaller amounts of lophenol from dietary cholesterol, desmosterol or sitosterol. When C. elegans was propagated in media containing sitosterol plus 25-azacoprostane hydrochloride (25-aza-5 beta-cholestane hydrochloride), an inhibitor of delta 24-sterol reductase in insects, its 4 alpha-methylsterol fraction largely consisted of equal amounts of 4 alpha-methyl-5 alpha-cholesta-7,24-dien-3 beta-ol and 4 alpha-methyl-5 alpha-cholesta-8(14),24-dien-3 beta-ol. Thus 25-azacoprostane hydrochloride inhibited both a delta 24-sterol reductase and a delta 7-sterol isomerase in C. elegans.

Cholesteryl oleate: Mounting sex pheromone of the hard tick Dermacentor variabilis (Say) (Acari: Ixodidae)☆

The moulting sex pheromone of Dermacentor variabilis, the American dog tick, is cholesteryl oleate. This steryl ester, when present in amounts from about 1 to 0.1 female equivalents elicited male mounting responses that were indistinguishable from the natural controls. Other steryl esters present on the ticks body surface also elicited responses, but a significantly lower intensity than cholesteryl oleate. Long regarded as a waste product of vertebrate blood feeding, this material is secreted by sexually mature females during feeding and accumulates on the surface of the body cuticle. Mate-seeking males, attracted to the females by the presence of 2,6-dichlorophenol, recognize and mount the feeding females. The accumulation of cholesteryl oleate on the females body surface during blood feeding is an indication of successful parasitism and acts as a signal of female mating readiness. Compounds of unknown composition that co-chromatographed with steryl esters were also found in fed females of 4 other ixodid tick species. Bioassays performed with crude extracts of these different species suggest that mounting sex pheromones are widespread in the Ixodidae and may also use the same or similar steryl esters, either alone or in mixtures, as in D. variabilis.

Catechins as germination and growth inhibitors in Lespedeza seeds

Abstract The germination and growth inhibiting compounds found in Lespedeza bicolor, L. cuneata and L. stipulacea seeds have been shown to be catechin and epicatechin. Differences in time of germination and amount of early seedling growth (L. stip. >L. cun. = L. bic.) were inversely related to the quantities of these polyphenols found in the seeds.