In vertebrates, symmetric versus asymmetric cleavage of -carotene in the biosynthesis of vitamin A and its derivatives has been controversially discussed. Recently we have been able to identify a cDNA encoding a metazoan ,-carotene-15,15-dioxygenase from the fruit fly Drosophila melanogaster. This enzyme catalyzes the key step in vitamin A biosynthesis, symmetrically cleaving -carotene to give two molecules of retinal. Mutations in the corresponding gene are known to lead to a blind, vitamin A-deficient phenotype. Orthologs of this enzyme have very recently been found also in vertebrates and molecularly characterized. Here we report the identification of a cDNA from mouse encoding a second type of carotene dioxygenase catalyzing exclusively the asymmetric oxidative cleavage of -carotene at the 9,10 double bond of -carotene and resulting in the formation of -apo-10-carotenal and -ionone, a substance known as a floral scent from roses, for example. Besides -carotene, lycopene is also oxidatively cleaved by the enzyme. The deduced amino acid sequence shares significant sequence identity with the ,-carotene-15,15-dioxygenases, and the two enzyme types have several conserved motifs. To establish its occurrence in different vertebrates, we then attempted and succeeded in cloning cDNAs encoding this new type of carotene dioxygenase from human and zebrafish as well. As regards their possible role, the apocarotenals formed by this enzyme may be the precursors for the biosynthesis of retinoic acid or exert unknown physiological effects. Thus, in contrast to Drosophila, in vertebrates both symmetric and asymmetric cleavage pathways exist for carotenes, revealing a greater complexity of carotene metabolism.
The xanthophylls lutein and zeaxanthin have attracted a lot of interest since it was presumed that an increased nutritional uptake may prevent adult macula degeneration (AMD). Although egg yolks serve as an important dietary source of lutein and zeaxanthin, data on xanthophyll concentrations in commercial egg yolks are not available. Thus, an high-performance liquid chromatography-diode array detector (HPLC-DAD) method was developed allowing for simultaneous separation of eight xanthophylls used to fortify poultry feed. Peak identification was carried out by liquid chromatography-atmospheric pressure chemical ionization mass spectrometry [LC-(APCI)MS]. Egg yolks of four types of husbandry (seven batches each) were examined. Lutein and zeaxanthin were the predominant xanthophylls in egg yolks produced in accordance with ecological husbandry (class 0) because the concentrations of these xanthophylls ranged from 1274 to 2478 microg/100 g and from 775 to 1288 microg/100 g, respectively. Analysis of variance (ANOVA) proved that both mean lutein and mean zeaxanthin concentrations of eggs from class 0 were statistically discriminable from mean lutein and zeaxanthin concentrations from eggs of all other classes (P < 0.01). Mean concentrations of synthetic xanthophylls in eggs of classes 1 (free range), 2 (barn), and 3 (cage) were as follows: canthaxanthin, 707 +/- 284 microg/100 g; beta-apo-8'-carotenoic acid ethyl ester, 639 +/- 391 microg/100 g; and citranaxanthin, 560 +/- 231 microg/100 g. Experiments with boiled eggs proved that beta-apo-8'-carotenoic acid ethyl ester was the xanthophyll with the highest stability, whereas lutein was degraded to the largest extent (loss of 19%). Detailed knowledge about the xanthophyll amounts in eggs is indispensable to calculate the human uptake.
Liquid chromatography-atmospheric pressure chemical ionization mass spectrometry (LC-APCIMS) was employed for the identification of eight lutein monoesters, formed by incomplete enzymatic saponification of lutein diesters of marigold (Tagetes erecta L.) by Candida rugosa lipase. Additionally, the main lutein diesters naturally occurring in marigold oleoresin were chromatographically separated and identified. The LC-MS method allows for characterization of lutein diesters occurring as minor components in several fruits; this was demonstrated by analysis of extracts of cape gooseberry (Physalis peruviana L.), kiwano (Cucumis metuliferus E. Mey. ex Naud.), and pumpkin (Cucurbita pepo L.). The assignment of the regioisomers of lutein monoesters is based on the characteristic fragmentation pattern: the most intense daughter ion generally results from the loss of the substituent (fatty acid or hydroxyl group) bound to the epsilon-ionone ring, yielding an allylic cation. The limit of detection was estimated at 0.5 microg/mL with lutein dimyristate as reference compound. This method provides a useful tool to obtain further insight into the biochemical reactions leading to lutein ester formation in plants.
It has been suggested that lutein and zeaxanthin may decrease the risk for age-related macular degeneration. Surprisingly, oleoresins rich in zeaxanthin are not yet available on the market. Several authors have reported enhanced stability of esterified xanthophylls, so plants containing zeaxanthin esters were investigated to establish valuable sources for the production of durable oleoresins. Liquid chromatography-atmospheric pressure chemical ionization mass spectrometry [LC-(APCI)MS] was used to unequivocally identify zeaxanthin esters of a standard mixture and in several plant extracts. Zeaxanthin esters were quantified on the basis of their respective molecular masses using zeaxanthin for calibration; total zeaxanthin was determined after saponification of aliquots of the extracts. Thus, dried wolfberries (Lycium barbarum), Chinese lanterns (Physalis alkekengi), orange pepper (Capsicum annuum), and sea buckthorn (Hippophae rhamnoides) proved to be valuable zeaxanthin ester sources. The present LC-MS method allows for an even more detailed analysis of zeaxanthin esters than reported previously.
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