β-Carotene, vitamin C and vitamin E content of the marine microalga Dunaliella tertiolecta cultured with different nitrogen sources

Variations in the β-carotene, vitamin C and vitamin E content of D. tertiolecta have been shown to result from the nitrogen source used in the culture medium. Differences of 101%, 38% and 69% have been found in β-carotene, ascorbic acid and tocopherol content in mg/g of dry matter, respectively, and differences of 147%, 63% and 37% occurred in β-carotene, vitamin C and E concentrations in mg/litre of culture, respectively. Considering the β-carotene, vitamin C and vitamin E content in mg/g of chlorophyll a, maximum variations occurred in β-carotene content, with differences of 145% among the different nitrogen sources. Maximum β-carotene and vitamin C values were found in urea cultures, whereas urea cultures showed the minimum values for vitamin E.     

The biotechnology of cultivating Dunaliella for production of β-carotene rich algae

Dunaliella accumulates massive amounts of β-carotene when cultivated under high light intensity and growth-limiting conditions. The pathway for biosynthesis of β-carotene was elucidated by analysis of the effect of selected inhibitors. The presence of the inhibitors elicited the accumulation of the following intermediates: β-zeacarotene, lycopene, ζ-carotene, phytofluene, phytoene and a few unidentified long-chain isoprenoids. Each of the accumulated intermediates was composed of about equal amounts of two stereisomers, as is the case for β-carotene in the untreated algae. It is deduced, therefore, that the isomerization reaction occurs early in the pathway of β-carotene biosynthesis, at or before phytoene.The unique caratenogenesis properties of Dunaliella led to the development of a new biotechnological process for mass-cultivation of the alga. Commercial production facilities for β-carotene rich Dunaliella exist today in Israel, USA, Australia, Spain and China. Recent developments, which indicate that the stereoisometric mixture of β-carotene present in Dunaliella is preferentially absorbed in animal tissues, coupled with new evidence for the efficacy of β-carotene in reducing the incidence of cancer, have opened new vistas of potential markets for the high β-carotene algae.     

Combinatorial expression of bacterial whole mevalonate pathway for the production of β-carotene in E. coli

The increased synthesis of building blocks of IPP (isopentenyl diphosphate) and DMAPP (dimethylallyl diphosphate) through metabolic engineering is a way to enhance the production of carotenoids. Using E. coli as a host, IPP and DMAPP supply can be increased significantly through the introduction of foreign MVA (mevalonate) pathway into it. The MVA pathway is split into two parts with the top and bottom portions supplying mevalonate from acetyl-CoA, and IPP and DMAPP from mevalonate, respectively. The bottom portions of MVA pathway from Streptococcus pneumonia, Enterococcus faecalis, Staphylococcus aureus, Streptococcus pyogenes and Saccharomyces cerevisiae were compared with exogenous mevalonate supplementation for β-carotene production in recombinant Escherichia coli harboring β-carotene synthesis genes. The E. coli harboring the bottom MVA pathway of S. pneumoniae produced the highest amount of β-carotene. The top portions of MVA pathway were also compared and the top MVA pathway of E. faecalis was found out to be the most efficient for mevalonate production in E. coli. The whole MVA pathway was constructed by combining the bottom and top portions of MVA pathway of S. pneumoniae and E. faecalis, respectively. The recombinant E. coli harboring the whole MVA pathway and β-carotene synthesis genes produced high amount of β-carotene even without exogenous mevalonate supplementation. When comparing various E. coli strains – MG1655, DH5α, S17-1, XL1-Blue and BL21 – the DH5α was found to be the best β-carotene producer. Using glycerol as the carbon source for β-carotene production was found to be superior to glucose, galactose, xylose and maltose. The recombinant E. coli DH5α harboring the whole MVA pathway and β-carotene synthesis genes produced β-carotene of 465 mg/L at glycerol concentration of 2% (w/v).     

Determination of lycopene, α-carotene and β-carotene in serum by liquid chromatography–atmospheric pressure chemical ionization mass spectrometry with selected-ion monitoring

A selected-ion monitoring (SIM) determination of serum lycopene, α-carotene and β-carotene by an atmospheric pressure chemical ionization mass spectrometry (APCI–MS) was developed. A large amount of serum cholesterols disturbed the SIM determination of carotenoids by contaminating the segment of interface with the LC–MS. Therefore, separation of carotenoids from the cholesterols was performed using a mixed solution of methanol and acetonitrile (70:30) as the mobile phase on a C₁₈ column of mightsil ODS-5 (75 mm×4.6 mm I.D.). The SIM determination was carried out by introducing only the peak portions of carotenoids and I.S. (squalene) by means of an auto switching valve. In the positive mode of APCI–MS, lycopene, α-carotene and β-carotene were monitored at m/z 537 and I.S. was monitored at m/z 411. This method was linear for all analytes in the range of 15–150 ng for lycopene, 7–70 ng for α-carotene and 25–50 ng for β-carotene. The detection limit of LC–APCI–MS-SIM for carotenoids was about 3 ng per 1 ml of serum (S/N=3). The repeatabilities, expressed as C.V.s, were 10%, 8.4% and 5.3% for lycopene, α-carotene and β-carotene, respectively. The intermediate precisions, expressed as C.V.s, were 11. 2%, 8.8% and 6.5% for lycopene, α-carotene and β-carotene, respectively.     

Lycopene-ε-Cyclase Pre-mRNA is Alternatively Spliced in Cara Cara Navel Orange (<Emphasis Type="Italic">Citrus sinensis</Emphasis> Osbeck)

Lycopene-ε-cyclase is one of the key enzymes related to α-carotene metabolism in plants. A full-length cDNA of 1300 bp encoding lycopene-ε-cyclase (Lyce) was generated from Cara Cara navel orange, a unique navel orange containing both lycopene and β-carotene in its pulp, with little or no α-carotene. The gene had a 14 bp nucleotides deletion and caused a terminal mutation. DNA sequence corresponding to the deletion region revealed that two repeats of 6 bp (AGGTGT) were flanking the region in both Cara Cara and its original variety, Washington navel oranges, but a 2 bp (AT) insertion was only found in Cara Cara which explain the alternative splicing character of the gene.     

Serum α- and γ-tocopherols, retinol, retinyl palmitate, and carotenoid concentrations in captive and free-ranging bottlenose dolphins (Tursiops truncatus)

Concentrations of retinol, retinyl palmitate, β-carotene, α-carotene, cryptoxanthin, lutein, lycopene, α-tocopherol, and γ-tocopherol were measured in blood samples collected from 15 captive and 55 free-ranging bottlenose dolphins (Tursiops truncatus). From June 1991 to June 1994, blood samples were collected from captive animals residing at two locations; at Seven Seas (Brookfield Zoo, Brookfield, IL) and Hawk’s Cay (Marathon Key, FL). Blood samples were collected from free-ranging animals from June 1991 to June 1996. Retinol levels were not significantly different between captive dolphin groups. However, Seven Seas animals had higher (P<0.01) serum retinol concentrations compared to free-ranging animals (0.061 vs 0.041 μg/ml). Retinyl palmitate was not detected in the serum of captive or free-ranging dolphins. Alpha-tocopherol levels were significantly (P<0.05) higher for Seven Seas dolphins (16.4 μg/ml) than for Hawk’s Cay (13.0 μg/ml) and free-ranging dolphins (12.5 μg/ml). Gamma-tocopherol concentrations were similar among captive and free-ranging dolphins. Free-ranging dolphins showed levels of circulating carotenoids (lutein and β-carotene) while the captive animals did not. Additional carotenoids (lycopene, α-carotene and cryptoxanthin) were analyzed but not detected in any samples. Serum vitamin differences between captive and free-ranging dolphins may reflect the natural diet or indicate some potential biological or nutritional status significance.     

Carotenoid discrimination by the avian embryo: a lesson from wild birds

The concentrations (μg/g wet yolk) of total carotenoids in eggs of the common moorhen (Gallinula chloropus), American coot (Fulica americana) and lesser black-backed gull (Larus fuscus), collected in the wild, were 47.5, 131.0 and 71.6, respectively. In contrast to data for eggs of the domestic chicken, β-carotene was a significant component in the yolks of these three wild species, forming 25–29% by wt. of the total carotenoids present. The concentration of total carotenoids in the livers of the newly-hatched chicks was 5–10 times higher than in the other tissues and β-carotene was again a major component, forming 37–58% of the hepatic carotenoids. In the newly-hatched gull, the proportions of both lutein and zeaxanthin were very low in the liver but high in the heart and muscle when compared with the yolk. By contrast canthaxanthin, echinenone and β-carotene were very minor constituents of heart and muscle when compared with their proportions in the yolk of the gull. The proportions of lutein and zeaxanthin in the liver of the newly-hatched coot and moorhen were also far lower than in the yolk whereas the liver was relatively enriched with β-cryptoxanthin, β-carotene and (in the moorhen) echinenone. The results indicate that avian embryos discriminate between different carotenoids during their distribution from the yolk to the various tissues.     

Characterization of the cytochrome <Emphasis Type="Italic">b</Emphasis><Subscript>6</Subscript><Emphasis Type="Italic">f</Emphasis> complex from marine green alga, <Emphasis Type="Italic">Bryopsis corticulans</Emphasis>

A pure, active cytochrome b ₆ f was isolated from the chloroplasts of the marine green alga, Bryopsis corticulans. To investigate and characterize this cytochrome b ₆ f complex, sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), absorption spectra measurement and HPLC were employed. It was shown that this purified complex contained four large subunits with apparent molecular masses of 34.8, 24, 18.7 and 16.7 kD. The ratio of Cyt b ₆ to Cytf was 2.01 : 1. The cytochromeb ₆ f was shown to catalyze the transfer of 73 electrons from decylplastoquinol to plastocyanin–ferricyanide per Cyt f per second. α-Carotene, one kind of carotenoid that has not been found to present in cytochrome b ₆ f complex, was discovered in this preparation by reversed phase HPLC. It was different from β-carotene usually found in cytochrome b ₆ f complex. The configuration of the major α-carotene component was assigned to be 9-cis by resonance Raman spectroscopy. Different from the previous reports, the configuration of this α-carotene in dissociated state was determined to be all-trans. Besides this carotene, chlorophyll a was also found in this complex. It was shown that the molecular ratios of chlorophylla, cis and all-trans-α-carotene to Cyt f in this complex were 1.2, 0.7 and 0.2, respectively.     

Astaxanthin accumulation in Haematococcus requires a cytochrome P450 hydroxylase and an active synthesis of fatty acids

Astaxanthin accumulation by green microalgae is a natural phenomenon known as red snows and blood rains. The fact that astaxanthin synthesis requires oxygen, NADPH and Fe²⁺ led Cunningham and Gantt [Annu. Rev. Plant Physiol. Plant Mol. Biol. 49 (1998) 557–583] to propose that a cytochrome P450-dependent enzyme might be involved in the transformation of β-carotene to astaxanthin. In Haematococcus only esterified astaxanthin molecules accumulate, but it is not determined whether a fatty acid synthesis should occur simultaneously to allow pigment accumulation. The aim of this contribution was to answer these two questions using specific inhibitors of β-carotene (norflurazon) and fatty acid (cerulenin) synthesis, and of cytochrome P450 enzyme activity (ellipticine).     

Ustilago maydis accumulates β-carotene at levels determined by a retinal-forming carotenoid oxygenase

The basidiomycete Ustilago maydis, the causative agent of corn smut disease, has emerged as a model organism for dimorphism and fungal phytopathogenicity. In this work, we line out the key conserved enzymes for β-carotene biosynthesis encoded by the U. maydis genome and show that this biotrophic fungus accumulates β-carotene. The amount of this pigment depended on culture pH and aeration but was not affected by light and was not increased by oxidative stress. Moreover, we identified the U. maydis gene, cco1, encoding a putative β-carotene cleavage oxygenase. Heterologous overexpression and in vitro analyses of purified enzyme demonstrated that Cco1 catalyzes the symmetrical cleavage of β-carotene to yield two molecules of retinal. Analyses of β-carotene and retinal contents in U. maydis cco1 deletion and over-expression strains confirmed the enzymatic function of Cco1, and revealed that Cco1 determines the β-carotene content. Our data indicate that carotenoid biosynthesis in U. maydis is carried out to provide retinal rather than to deliver protective pigments. The U. maydis genome also encodes three potential opsins, a family of photoactive proteins that use retinal as chromophore. Two opsin genes showed different light-regulated expression patterns, suggesting specialized roles in photobiology, while no mRNA was detected for the third opsin gene in the same experiments. However, deletion of the cco1 gene, which should abolish function of all the retinal-dependent opsins, did not affect growth, morphology or pathogenicity, suggesting that retinal and opsin proteins play no relevant role in U. maydis under the tested conditions.     

The extensive commercial cultivation of Dunaliella salina

Betatene Ltd has established an extensive system for cultivation of D. salina for β-carotene production at Whyalla in South Australia.The operation, believed to be the largest area under man-made algal cultivation in the world, covers over 300 hectares in area, divided into three major cultivation lakes. A necessary requirement of the extensive system is to have a cost-effective harvesting system to handle the relatively large culture volumes. The extraction plant at the lakeside is designed to handle up to one million litre per hour in four parallel modules using a proprietary purely physical harvesting system.Current products are a crystalline β-carotene preparation for food colour applications, a high solubility preparation dispersed in soya bean oil for dietary supplementation and a dried powdered algal extract as an animal feed ingredient.     

Effect of Carotene and Lycopene on the Risk of Prostate Cancer: A Systematic Review and Dose-Response Meta-Analysis of Observational Studies

Background

Many epidemiologic studies have investigated the association between carotenoids intake and risk of Prostate cancer (PCa). However, results have been inconclusive.

Methods

We conducted a systematic review and dose-response meta-analysis of dietary intake or blood concentrations of carotenoids in relation to PCa risk. We summarized the data from 34 eligible studies (10 cohort, 11 nested case-control and 13 case-control studies) and estimated summary Risk Ratios (RRs) and 95% confidence intervals (CIs) using random-effects models.

Results

Neither dietary β-carotene intake nor its blood levels was associated with reduced PCa risk. Dietary α-carotene intake and lycopene consumption (both dietary intake and its blood levels) were all associated with reduced risk of PCa (RR for dietary α-carotene intake: 0.87, 95%CI: 0.76–0.99; RR for dietary lycopene intake: 0.86, 95%CI: 0.75–0.98; RR for blood lycopene levels: 0.81, 95%CI: 0.69–0.96). However, neither blood α-carotene levels nor blood lycopene levels could reduce the risk of advanced PCa. Dose-response analysis indicated that risk of PCa was reduced by 2% per 0.2mg/day (95%CI: 0.96–0.99) increment of dietary α-carotene intake or 3% per 1mg/day (95%CI: 0.94–0.99) increment of dietary lycopene intake.

Conclusions

α-carotene and lycopene, but not β-carotene, were inversely associated with the risk of PCa. However, both α-carotene and lycopene could not lower the risk of advanced PCa.     

Interference in Carotenogenesis as a Mechanism of Action of the Pyridazinone Herbicide Sandoz 6706: Accumulation of C-40 Carotenoid Precursors Inhibition of beta-Carotene Synthesis and Enhancement of Phytoene Epoxidation

The herbicide Sandoz 6706 (4-chloro-5-(dimethylamino)-2-α,α,α, (trifluoro-m-tolyl)-3(2H)-pyridazinone), when applied as a preplant soil treatment at a concentration of 0.05 μg/g reduced the content of β-carotene and chlorophylls in 21-day-old wheat seedlings (Triticum aestivum L.) by 55% and 29%, respectively, without affecting the fresh or dry matter of the seedlings. At 0.8 μg/g, the herbicide reduced the content of β-carotene and chlorophyll by as much as 98%, while the fresh weight of the albino seedlings was reduced by only 24%. The effect of the herbicide on chlorophyll b was much stronger than on chlorophyll a. Time course studies of pigment synthesis in Sandoz 6706-treated seedlings showed that chlorophyll, β-carotene, cyclic xanthophylls, phytoene, phytofluene, and ζ-carotene were accumulating during the first 7 days after sowing. Later on, there was a sharp decline in the content of chlorophyll and β-carotene and a gradual reduction in the content of phytofluene, ζ-carotene, and cyclic xanthophylls; the content of phytoene remained essentially unchanged. Coinciding with the drop in content of β-carotene and chlorophyll, there was a remarkable increase in the content of epoxy phytoene. It is suggested that Sandoz 6706 might act as an inhibitor of the cyclization reaction in the biosynthetic pathway of carotenoids and that other effects, such as the bleaching of chlorophyll, are a consequence of this inhibition.     

Simultaneous determination of retinol, β-carotene and α-tocopherol in adipose tissue by high-performance liquid chromatography

A method is described for evaluation of fat-soluble vitamin in human adipose tissue with the aim to obtain, accurately and within the shortest analysis time, a time-integrated measure of exposure to vitamins from the diet. Fat tissue was deproteinized with ethanol and extracted with n-hexane. Normal-phase HPLC was performed in a Lichrosorb Si60 column with a gradient of n-hexane–2-propanol at 1 ml/min. Detection was accomplished using a diode-array system (for retinol and β-carotene) in series with a fluorescence detector (α-tocopherol). The method was validated and applied to human adipose tissue in a total of 140 subjects. The mean contents found were 0.43, 0.84, 240.3 μg/g for retinol, β-carotene and α-tocopherol, respectively. The method is sensitive enough for detecting the compounds in 1.6 mg of adipose tissue considering the lowest concentration found.     

Main features of the oxidative metabolism in gills and liver of Odontesthes nigricans Richardson (Pisces, Atherinopsidae)

The aim of this work was to study comparatively the oxidative metabolism in gills and liver of a silverside, Odontesthes nigricans, in their natural environment, the Beagle Channel. Oxidative damage to lipids was evaluated by assessing TBARS and lipid radical content, in gills and liver. Gills showed a significantly higher degree of damage than liver. The content of α-tocopherol, β-carotene and catalase activity showed significantly higher values in the liver than in the gills. The ascorbyl radical (A^•) content showed no significant differences between gills and liver. The ascorbate (AH⁻) content was 12 ± 2 and 159 ± 28 nmol/mg FW in gills and liver, respectively. Oxidative metabolism at the hydrophilic level was assessed as the ratio A^•/AH⁻. The ratio A^•/AH⁻ was significantly different between organs, (6 ± 2)10^− 5 and (5 ± 2)10^− 6, for the gills and the liver, respectively. Both, lipid radical content/α-tocopherol content and lipid radical content/β-carotene content ratios were significantly higher in gills as compared to the values recorded for the liver, suggesting an increased situation of oxidative stress condition in the lipid phase of the gills. Taken as a whole, the O. nigricans liver exhibited a better control of oxidative damage than the gills, allowing minimization of intracellular damage when exposed to environmental stressing conditions.     

Stability of individual carotenoids, retinol and tocopherols in human plasma during exposure to light and after extraction

We have modified gradient HPLC procedures for simultaneous quantification of retinol, γ-tocopherol, α-tocopherol, lutein/zeaxanthin, β-cryptoxanthin, trans-lycopene, cis-lycopene, α-carotene and β-carotene in 200-μl aliquots of human plasma. The photosensitivity of these analytes in plasma exposed to fluorescent lighting for up to 72 h was investigated and most were stable under these conditions. The stability of these analytes held in darkness at −20°C, 4°C or room temperature for up to 48 h after extraction from plasma was also investigated. Variability in measurement of most analytes was greater at room temperature than at 4°C or −20°C. There were statistically significant variations in the measured concentrations of some analytes in samples kept cold. However, the magnitude of these variations was small and of little biological significance, particularly over the first 24 h.     

Occurrence of 9′Z-β-Echinenone in the Sea Urchin Pseudocentrotus depressus

β-Echinenone is a major carotenoid in the gonad of sea urchins and may play an important role in reproduction and embryonic development. We reinvestigated β-echinenone occurrence in the gonad, viscera, test, and spine of the sea urchin Pseudocentrotus depressus. It was found that β-echinenone fraction consisted of all-E- and 9′Z-β-echinenone. The highest abundance of 9′Z-β-echinenone (76.0–78.2% of the total β-echinenone fraction) was observed in the ovary and testis of the sea urchin. In both females and males, all-E-β-echinenone predominated in the viscera (63.6–75.9%), unlike the 9′Z-β-echinenone, and it was also present in the test and spine (41.3–64.9%). It should be made clear that the work suggests that the Z-carotenoid may have a specific function in the sea urchin, possibly related to reproduction.     

Conversion of carotenoids into vitamins A(1) and A(2) in two species of freshwater fish

1. Examination of two zooplankton species predominating in fish ponds, Daphnia magna and Chironomus larvae, revealed the presence of α- and β-carotene, echinenone, canthaxanthin and 3-hydroxy-4-oxo-β-carotene in Daphnia, and β-carotene and cryptoxanthin ester in Chironomus. No specific provitamins A₂ (containing a 3,4-dehydro-β-ionone ring) were detected. 2. Guppies (Lebistes reticulatus) and platies (Xiphophorus variatus) were found to form vitamin A from β-carotene and from its oxygen-containing derivatives isozeaxanthin, canthaxanthin and astaxanthin. Slight conversion into vitamin A₂ seemed to occur simultaneously. 3,4-Dehydro-3′-hydroxy-β-carotene formed little vitamin A, and the latter was mainly of the A₂ type. Lutein was devoid of provitamin A properties. 3. In addition to vitamin A, β-carotene was detected in fish receiving the 4-oxo- and 4-hydroxy-carotenoids. A reaction scheme for the conversion of carotenoids into retinal and and 3,4-dehydroretinal is presented. 4. It is concluded that natural 4-oxo derivatives of β-carotene may play a significant role as vitamin A precursors for fish.