Carotenoids in fish. XXVI. Pungitius pungitius (L.) and Gasterosteus aculeatus L. (Gasterosteidae)

The author investigated the presence of various carotenoids in the different parts of the body of Pungitius pungitius (L.) and Gasterosteus aculeatus L. by means of columnar and thin-layer chromatography. The investigations revealed the presence of the following carotenoids:

in Pungitius pungitius. α-carotene, β-carotene, β-cryptoxanthin, mutatochrome, zeaxanthin and astaxanthin;

in Gasterosteus aculeatus: β-carotene, β-cryptoxanthin, β-carotene epoxide, neothxanthin, canthaxanthin, mutatochrome, lutein, phoenicoxanthin, zeaxanthin, taraxanthin, tunaxanthin, astaxanthin, astaxanthin ester and α-doradexanthin. The total carotenoid content ranged from 2.229 to 138.504 µg/g wet weight.

     

Genetic analysis of provitamin A carotenoid β-cryptoxanthin concentration and relationship with other carotenoids in maize grain (<Emphasis Type="Italic">Zea mays</Emphasis> L<Emphasis Type="Italic">.</Emphasis>)

Provitamin A (proVA) carotenoids are converted into retinol (vitamin A) in the human body, are the subject of human nutrition studies, and are targets for biofortification of staple crops. β-Carotene has been the principal target for enhancing levels of proVA. There is recent interest in enhancing the proVA carotenoid β-cryptoxanthin since it has excellent bioavailability, and in maize may be nearly as effective as β-carotene in providing retinol to humans. This study was designed to enhance our understanding of the genetic control of: levels of β-cryptoxanthin, conversion of β-carotene into β-cryptoxanthin and zeaxanthin, conversion of β-cryptoxanthin into zeaxanthin, and flux into and within the β-branch of carotenoid pathway. A biparental population derived from two inbreds with relatively high levels of β-cryptoxanthin and different ratios of β-carotene to β-cryptoxanthin and β-cryptoxanthin to zeaxanthin was studied. Three field replications of this F_2:3 population were grown, grain analyzed by liquid chromatography (LC), and composite interval mapping (CIM) performed to identify 90 quantitative trait loci (QTL) for carotenoids. We detected QTL for β-carotene/(β-cryptoxanthin + zeaxanthin) and (β-carotene + β-cryptoxanthin)/zeaxanthin ratios that contain candidate gene hydroxylase 4 (hyd4), which has not been previously associated with QTL for carotenoids in maize grain. Two color assessment methods, visual score and chromameter reading, were used to phenotype one replicate of the population for initial assessment as simple alternative measuring procedures. A common finding for LC and chromameter analysis included QTL on chromosome 5 that contain candidate gene lycopene β cyclase (lcyβ).     

In vitro and in vivo biosynthesis of xanthophylls by the cyanobacterium Aphanocapsa

Of the six carotenoids identified in the cyanobacterium Aphanocapsa, β-carotene, zeaxanthin, echinenone and myxoxanthophyll are the major pigments, whilst β-cryptoxanthin and 3-hydroxy-4-keto-β-carotene are present only in trace amounts. With the exception of zeaxanthin, the other xanthophylls could be formed in vitro from [¹⁴C]phytoene in high yields, especially β-cryptoxanthin and 3-hydroxy-4-keto-β-carotene. In a time course experiment of xanthopyll biosynthesis the flow of radioactivity from [¹⁴C]phytoene was followed through the pools of phytofluene, lycopene, and β-carotene. The reaction sequence from phytoene to xanthophylls is sensitive in vitro to both difunone, an inhibitor of carotene desaturation, and CPTA, an inhibitor of cyclization.     

Plasma tocopherol,retinol, and carotenoid concentrations in free-ranging Humboldt penguins (Spheniscus humboldti) in Chile

Plasma retinol and α-tocopherol concentrations were measured in heparinized blood samples collected from 51 free-ranging adult Humboldt penguins (Sphenicus humboldti) residing at two colonies off the Chilean coast. Thirty samples were collected in April 1992 from penguins inhabiting the Ex-islote de los Pájaros Niños in Algarrobo, Chile. In September 1992, 21 samples were collected from birds inhabiting Isla de Cachagua, Chile. Samples were assayed for retinol, retinyl palmitate, α-tocopherol, γ-tocopherol, lutein, β-cryptoxanthin, lycopene, α-carotene, and β-carotene. Retinol, α-tocopherol, and lutein were detected in all samples, while lycopene and γ-tocopherol were not detected in any. A significantly higher percentage of samples had detectable levels of retinyl palmitate and α-carotene in April (P < 0.001): for β-cryptoxanthin the percentage was higher in September (P < 0.001). Plasma concentrations of α-tocopherol and lutein were higher in September. Alpha-tocopherol concentrations were 1,877.1 ± 99.0 (SEM) μg/dl in April compared to 2.289 ± 122.3 μg/dl in September (P < 0.05); lutein concentrations were 4.16 ± 0.43 μg/dl in April vs. 10.68 ± 1.02 μg/dl in September (P < 0.001). Retinol concentrations were not significantly different (117 ± 8.0 μg/dl in April vs. 105.3 ± 7.6 μg/dl in September). Both physiologic changes associated with season, and the change in locale may have contributed to the differences seen in the assay means and the number of samples with detectable levels. © 1996 Wiley-Liss, Inc.     

Over-expression of Arabidopsis thaliana carotenoid hydroxylases individually and in combination with a β-carotene ketolase provides insight into in vivo functions

Carotenoids represent a group of widely distributed pigments derived from the general isoprenoid biosynthetic pathway that possess diverse functions in plant primary and secondary metabolism. Modification of α- and β-carotene backbones depends in part on ring hydroxylation. Two ferredoxin-dependent non-heme di-iron monooxygenases (AtB1 and AtB2) that mainly catalyze in vivo β-carotene hydroxylations of β,β-carotenoids, and two heme-containing cytochrome P450 (CYP) monooxygenases (CYP97A3 and CYP97C1) that preferentially hydroxylate the ε-ring of α-carotene or the β-ring of β,ε-carotenoids, have been characterized in Arabidopsis by analysis of loss-of-function mutant phenotypes. We further investigated functional roles of both hydroxylase classes in modification of the β- and ε-rings of α-carotene and β-carotene through over-expression of AtB1, CYP97A3, CYP97C1, and the hydroxylase candidate CYP97B3. Since carotenoid hydroxylation is required for generation of ketocarotenoids by the bkt1(CrtO) β-carotene ketolase, all hydroxylase constructs were also introduced into an Arabidopsis line expressing the Haematococcus pluvalis bkt1 β-carotene ketolase. Analysis of foliar carotenoid profiles in lines overexpressing the individual hydroxylases indicate a role for CYP97B3 in carotenoid biosynthesis, confirm and extend previous findings of hydroxylase activities based on knock-out mutants, and suggest functions of the multifunctional enzymes in carotenoid biosynthesis. Hydroxylase over-expression in combination with bkt1 did not result in ketocarotenoid accumulation, but instead unexpected patterns of α-carotene derivatives, accompanied by a reduction of α-carotene, were observed. These data suggest possible interactions between the β-carotene ketolase bkt1 and the hydroxylases that impact partitioning of carbon flux into different carotenoid branch pathways.     

Carotenoids in red algae

The carotenoid composition of the following 8 species of red algae has been studied quantitatively and qualitatively: Bangia fuscopurpurea, Nemalion helminthoides, Bonnemaisonia hamifera (tetrasporophyte), Gigartina stellata, Rhodymenia palmata, Ceramium rubrum, Polysiphonia brodiaei, and Polysiphonia urceolata. Naturally occurring material of G. stellata, R. palmata, and P. brodiaei was investigated, while monoalgally cultured material was obtained from the remaining 5 species. α- and β-carotene, lutein, zeaxanthin, and small amounts of α- or β-cryptoxanthin were commonly present, but the two species P. brodiaei and P. urceolata were devoid of carotenoids containing α-ionone rings. Fucoxanthin was detected in naturally occurring material of B. fuscopurpurea, N. helminthoides, C. rubrum, and P. brodiaei, but could not be found in cultured material of the 3 first-mentioned species. The possible origin of the fucoxanthin is discussed as well as the biochemical and phylogenetic implications of the results obtained. As judged from the electronic spectrum of the total extracts, chlorophyll d was not present in any of the algae investigated. The total amounts of carotenoids and the ratio carotenoids: chlorophyll a were similar to those reported in previous work.     

Effect of light intensity on the formation of carotenoids in normal and mutant maize leaves

Carotenoid composition in leaves of normal, lycopenic and ζ-carotenic mutants of Zea mays were investigated. In lycopenic leaves, in addition to lycopene, phytoene, phytofluene, δ- and γ-carotene, trace amounts of α- and β-carotene and antheraxanthin were identified. Low light promoted accumulation of α- and β-carotene; high light brought about an increase in antheraxanthin content. In the leaves of the ζ-carotenic mutant, phytoene, phytofluene and ζ-carotene were synthesized. Illumination of low intensity stimulated carotenoid synthesis to a slight extent. Relative amounts of carotenoid components were essentially the same as in etiolated material, except for a small increase in cis-ζ-carotene. Under high intensity illumination, carotenoids were rapidly destroyed.     

Effects of single and mixed inoculation with two arbuscular mycorrhizal fungi in two different levels of phosphorus supply on β-carotene concentrations in sweet potato (Ipomoea batatas L.) tubers

Aims

This study aimed to determine the effect of arbuscular mycorrhizal (AM) fungi and phosphorus (P) supply levels on β-carotene concentrations in sweet potato (Ipomoea batatas L.) tubers.

Methods

Two commercial AM fungal isolates of Glomus intraradices (IFP Glintra) and Glomus mosseae (IFP Glm) which differ in their life cycles were used. Sweet potato plants were grown in a horizontal split-root system that consisted of two root compartments. A root-free fungal compartment that allowed the quantification of mycelial development was inserted into each root compartment. The two root compartments were inoculated either with the same or with different AM isolates, or remained free of mycorrhizal propagules. Each fungal treatment was carried out in two P supply levels.

Results

In the low P supply level, mycorrhizal colonization significantly increased β-carotene concentrations in sweet potato tubers compared with the non-mycorrhizal plants. Glomus intraradices appeared to be more efficient in increasing β-carotene concentrations than G. mosseae. Dual inoculation of the root system with the two mycorrhizal fungi did not result in a higher increase in tuber β-carotene concentrations than inoculation with the single isolates. Improved P nutrition led to higher plant tuber biomass but was not associated with increased β-carotene concentrations.

Conclusions

The results indicate a remarkable potential of mycorrhizal fungi to improve β-carotene concentrations in sweet potato tubers in low P fertilized soils. These results also suggest that β-carotene metabolism in sweet potato tubers might be specifically activated by root mycorrhizal colonization.     

Isocratic rapid liquid chromatographic method for simultaneous determination of carotenoids,retinol, and tocopherols in human serum

An improved isocratic and rapid HPLC method was developed for the measurement of carotenoids, retinol and tocopherols in human serum. Vitamins were extracted with hexane. Mobile phase consisted of a mixture acetonitrile:methylene chloride:methanol with 20 mM ammonium acetate. This method used a small bead size (3 μm) Spherisorb ODS2 column with titane frits. Diode array and fluorescence detectors were used respectively for the detection of carotenoids and retinol/tocopherols. Chromatographic separation was complete in 13 min for β-cryptoxanthin, cis–trans-lycopene, α-carotene, β-carotene, cis-β-carotene, retinol, δ-tocopherol, γ-tocopherol and α-tocopherol. Echinenone and tocol were employed as internal standards for diode array and fluorescence detection. Accuracy was validated using standard reference material (SRM) 968C. Intra-assay and inter-assay precision were respectively 0.2–7.3% and 3.6–12.6%. Sensitivity was verified using the ICH recommendations and the limit of detection (LOD) obtained was sufficient for routine clinical application.     

Carotenoids fromCapsicum annuum fruits: Changes during ripening and storage

Contents of individual carotenoids in the fruits of Ramillete cultivar ofCapsicum annuum L. changed during ripening of recently harvested fruits or fruits stored 15 d in a dark and cool room. β-carotene and β-cryptoxanthin contents rose to a maximum and then declined, while the contents of other earotenoids rose to a rather steady level. Capsanthin was the main carotenoid which together with capsorubin were responsihle for the ripening colour.     

β-Carotene as a high-potency antioxidant to prevent the formation of phospholipid hydroperoxides in red blood cells of mice

In order to investigate the antioxidant effect of β-carotene in vivo, phospholipid hydroperoxides and β-carotene isomers in red blood cells (RBC), plasma and tissue organelles were quantitatively measured after the oral administration of β-carotene (94.8% all-trans-β-carotene) to mice. Three groups of 24 mice each were fed for 1 week on a semisynthetic diet supplemented with either 0.6% or 3.0% β-carotene/diet or maintained on a control (β-carotene-unsupplemented) diet. The RBC phospholipid hydroperoxides showed a significant decrease followed by an increase of β-carotene intakes; i.e., 201, 16 and 4 pmol of phosphatidylcholine hydroperoxide/ml packed RBC, and 108, 22 and 8 pmol of phosphatidylethanolamine hydroperoxide/ml packed RBC, in the mice given the control diet, 0.6% carotene diet and 3.0% carotene diet, respectively. The RBC β-carotene increased from 14 to 43 pmol/ml packed RBC as followed by the increase of β-carotene intakes. Such a potent antioxidant effect of β-carotene as observed in RBC was not confirmed in the plasma, liver or lungs, although their β-carotene contents increased. The β-carotene ingestion increased the all-trans-β-carotene d and retinol contents in RBC, plasma, liver and lungs, but the α-tocopherol content decreased. In the β-carotene-supplemented (6 g and 30 g/kg diet) mice, cis-β-carotene content was relatively higher in the RBC (25–35% of total β-carotene) than that in plasma, liver and lungs. The present findings indicate that not only does β-carotene act as a potent antioxidant in vivo but also its antioxidant effect is very specific in the RBC phospholipid bilayers rather than in the plasma and other tissue organelles.     

Heterologous biosynthesis of lutein in S. cerevisiae enabled by temporospatial pathway control

The market-expanding lutein is currently mainly supplied by plant extraction, with microbial fermentation using engineered cell factory emerging as a promising substitution. During construction of lutein-producing yeast, α-carotene formation through asymmetric ε- and β-cyclization of lycopene was found as the main limiting step, attributed to intra-pathway competition of the cyclases for lycopene, forming β-carotene instead. To solve this problem, temperature-responsive expression of β-cyclase was coupled to constitutive expression of ε-cyclase for flux redirection to α-carotene by allowing ε-cyclization to occur first. Meanwhile, the ε-cyclase was engineered and re-localized to the plasma membrane for further flux reinforcement towards α-carotene. Finally, pathway extension with proper combination of carotenoid hydroxylases enabled lutein (438 μg/g dry cells) biosynthesis in S. cerevisiae. The success of heterologous lutein biosynthesis in yeast suggested temporospatial pathway control as a potential strategy in solving intra-pathway competitions, and may also be applicable for promoting the biosynthesis of other natural products.     

Carotenoids in sixty-six representatives of the Pteridophyta

The presence of 27 carotenoids was determined in the Pteridophyta. The carotenoids characteristic of club-moss and horsetail species are β-carotene, β-cryptoxanthin, lutein epoxide and zeaxanthin, and fern species are β-cryptoxanthin, lutein epoxide, zeaxanthin, violaxanthin and rhodoxanthin.     

Effect of α-Tocopherol, β-Carotene, Monogalactosyldiglyceride and Phosphatidylcholine on Light-Induced Degradation of Chlorophyll a in Acetone

The effect of α-tocopherol, β-carotene, monogalactosyldi-glyceride and phosphatidylcholine on red light induced degradation of chlorophyll a was studied in acetone at 4°C. Monogalaclosyldi-glyceride was ineffective up to a molar ratio of monogalactosyldi glyceride to chlorophyll of 1:10. α-Tocopherol, β-carotene and phosphatidylcholine inhibited chlorophyll degradation. Maximal protection by α tocopherol and β-carotene was similar (76%) but on a molar basis a tocopherol was less effective. Protection by phosphatidylcholine was less than by a tocopherol and α-carotene but the lipid was effective at a lower ratio of chlorophyll to protectant. Inhibition by phosphatidylcholine was independent of the degree of unsaturation of the fatty acids. Effects of β-carotene and α-tocopherol were additive at suboptimal concentrations, but addition did not increase the maximal protection of 76% by these substances alone. Phosphatidylcholine increased the effectiveness of α-tocopherol and β-carotene independent of their concentrations. It is suggested that interactions between lipids participate in the mechanism protecting chlorophyll a against photooxidation in the chloroplast membrane.     

Engineering central metabolic modules of Escherichia coli for improving β-carotene production

ATP and NADPH are two important cofactors for production of terpenoids compounds. Here we have constructed and optimized β-carotene synthetic pathway in Escherichia coli, followed by engineering central metabolic modules to increase ATP and NADPH supplies for improving β-carotene production. The whole β-carotene synthetic pathway was divided into five modules. Engineering MEP module resulted in 3.5-fold increase of β-carotene yield, while engineering β-carotene synthesis module resulted in another 3.4-fold increase. The best β-carotene yield increased 21%, 17% and 39% after modulating single gene of ATP synthesis, pentose phosphate and TCA modules, respectively. Combined engineering of TCA and PPP modules had a synergistic effect on improving β-carotene yield, leading to 64% increase of β-carotene yield over a high producing parental strain. Fed-batch fermentation of the best strain CAR005 was performed, which produced 2.1 g/L β-carotene with a yield of 60 mg/g DCW.     

An in silico study of the citrus dioxygenases CCD4 family substrates

The coloration of Citrus fruits is related with the concentration of carotenoids, isoprenoid pigments of 40 carbon atoms (C₄₀). Rodrigo et al. and Ma et al. reported a CCD4-type citrus dioxygenase responsible for the generation of C₃₀ apocarotenoids providing a reddish-orange pigmentation to the peel of many mandarins and oranges. Among them, CCD4b was the first case described of a dioxygenase that cleaves carotenoids C₄₀ in the double bond 7′, 8′ or 7, 8, generating β-citraurin or 8-β-apocarotenal. Here we report the three-dimensional structures of CCD4a and CCD4b, modeled by sequence homology (2BIW) and validated by molecular dynamics (MD). Docking calculations were performed in CCD4a and CCD4b structures with thousands of rotated initial carotenoid conformations and all the possible poses in the active site were found. The interaction energy was measured by means of ASE scoring, Amber99 refinement and London ΔG rescoring. For the case of CCD4a model, the results showed London ΔG score of ?19, ?17 and ?15?kcal/mol for zeaxanthin, β-cryptoxanthin and β-carotene, respectively. The same sequence in the estimated interaction strength for the three ligands was obtained using MD. The interaction energy of CCD4b indicated that, in agreement with experimental data, zeaxanthin and β-cryptoxanthin could be cleaved by the enzyme, β- and α-carotene have chances to be oxidized and lycopene has not good interaction energy to be predicted as substrate. These findings will be discussed considering the potential in vivo substrates and products, and the physiological role in Citrus fruits.

Communicated by Ramaswamy H. Sarma     

Antioxidant activity of palm oil carotenes in peroxyl radical-mediatedperoxidation of phosphatidyl choline liposomes

Abstract

The antioxidant efficacy of α-carotene and comparison with β-carotene in multilamellar liposomes prepared from egg yolk phosphatidyl choline (EYPC) exposed to the lipid soluble 2,2′-azobis (2,4-dimethyl valeronitrile) (AMVN) was investigated. Lipid peroxidation was measured as thiobarbituric acid reacting substances (TBARS)at 532 nm or as hydroperoxide formation at 234 nm after separation of phosphatidyl choline hydroperoxide (PCOOH) by high-pressure liquid chromatography (HPLC). Lutein and zeaxanthin, the hydroxyl derivatives of α- and β-carotenes, and the chain breaking antioxidant α-tocopherol were also included in the study.AMVN being a lipid soluble, non polar azo initiator penetrates into the hydrophobic interior of the phospholipid bilayer, forming peroxyl radicals which peroxidate the phospholipid leading to PCOOH accumulation. All the carotenoids tested at 1 mol% relative to EYPC significantly suppressed the formation of PCOOH compared to control samples.In this system, α-carotene retarded PCOOH formation better than β-carotene. Similarly, lutein was a better antioxidant than is zeaxanthin. But lutein and zeaxanthin were more effective antioxidants than α- and β-carotenes, respectively. After 1 h of incubation of the carotenoid with AMVN, α-, β-carotene, lutein and zeaxanthin limited PCOOH formation by 77%, 68%, 85%and 82%, respectively, while α-tocopherol elicited 90%reduction.AMVN incubated with EYPC for 2 h induced the formation of TBARS compared to control (P <0.001). α-Carotene significantly suppressed the TBARS formation by 78% whilst β-carotene, lutein, zeaxanthin and α-tocopherol elicited 60%, 91%and 80% reductions, respectively. Increasing the concentration of the carotenoid >1 mol% to EYPC did not significantly increase protection of the membrane against free radical attack.Our findings suggest that α-carotene is a better antioxidant than is β-carotene in phosphatidyl choline vesicles. It may, therefore, be useful in limiting free radical mediated peroxidative damage against membrane phospholipids _{in vivo}.