beta-carotene and astaxanthine

Carotenoids are natural pigments that widely exist in nature. Due to their excellent antioxidant, anticancer and anti-inflammatory properties, carotenoids are commonly used in food, medicine, cosmetic and other fields. At present, natural carotenoids are mainly extracted from plants, algae and microorganisms. With the rapid development of metabolic engineering and molecular biology as well as the continuous in-depth study of carotenoids synthesis pathways, industrial microorganisms have showed promising applications in the synthesis of carotenoids. In this review, we introduced the properties of several carotenoids and their biosynthetic metabolism process. Then, the microorganisms synthesizing carotenoids through the natural and non-natural pathways and the extraction methods of carotenoids were summarized and compared. Meanwhile, the influence of substrates on the carotenoids production was also listed. The methods and strategies for achieving high carotenoid production are categorized to help with future research.

Topics: beta Carotene; Carotenoids; Lycopene; Xanthophylls

The current review provides an up-to-date analysis of scientific data on astaxanthin (ASX) sources and experimental studies on its health benefits as a potent antioxidant in the aging process. ASX is a liposoluble carotenoid nutrient and reddish-orange pigment, naturally synthesized by numerous microalgae, yeasts, and bacteria as secondary metabolites. Provides a reddish hue to redfish and shellfish flesh that feed on ASX-producing microorganisms. The microalga

Topics: Antioxidants; beta Carotene; Dietary Supplements; Xanthophylls

Astaxanthin is a natural pigment, known for its strong antioxidant activity and numerous health benefits to human and animals. Its antioxidant activity is known to be substantially greater than β-carotene and about a thousand times more effective than vitamin E. The potential health benefits have generated a growing commercial interest, and the escalating demand has prompted the exploration of alternative supply chain. Astaxanthin naturally occurs in many sea creatures such as trout, shrimp, and microalgae, some fungi, bacteria, and flowering plants, acting to protect hosts against environmental stress and adverse conditions. Due to the rapid growth and simple growth medium requirement, microbes, such as the microalga, Haematococcus pluvialis, and the fungus Xanthophyllomyces dendrorhous, have been developed to produce astaxanthin. With advances in metabolic engineering, non-carotenogenic microbes, such as Escherichia coli and Saccharomyces cerevisiae, have been purposed to produce astaxanthin and significant progress has been achieved. Here, we review the recent achievements in microbial astaxanthin biosynthesis (with reference to metabolic engineering strategies) and extraction methods, current challenges (technical and regulatory), and commercialization outlook. Due to greenness, sustainability, and dramatic cost reduction, we envision microbial synthesis of astaxanthin offers an alternative means of production (e.g. chemical synthesis) in the near future.

Topics: Bacteria; beta Carotene; Bioreactors; Biosynthetic Pathways; Fungi; Metabolic Engineering; Microalgae; Xanthophylls

Astaxanthin and β-carotene are important carotenoids used in numerous pharmaceutical and nutraceutical applications, owing to their vigorous antioxidant properties. The microalgal strains Haematococcus pluvialis and Dunaliella salina accumulate the highest quantities of astaxanthin and β-carotene (up to 7% and 13% dry weight respectively) and are therefore considered as sustainable feedstock for the commercial production of carotenoids. Thus, from an economical perspective, it becomes desirable to optimize recovery of carotenoids from microalgal cells. To this end, here, we have summarized the conventional and modern extraction techniques generally used for the recovery of astaxanthin from Haematococcus pluvialis and β-carotene from Dunaliella salina. Furthermore, we have also discussed the optimum process conditions employed for numerous extraction protocols including solvent extraction, ultrasonic-assisted extraction (UAE), microwave-assisted extraction (MAE) and supercritical fluid extraction (SFE). Overall, our study highlights the sustainability of integrated co-production of biofuels and carotenoids in a biorefinery framework.

Topics: beta Carotene; Chemical Fractionation; Chlorophyceae; Xanthophylls

Carotenoids relevance as natural pigments is mainly due to their uses as colorants, feed supplements, nutraceuticals and for medical, cosmetic, and biotechnological purposes. Since they have putative health beneficial effects, the demand and market of carotenoids are growing significantly. There is a diversity of natural and synthetic carotenoids, but only a few of them are commercially produced, including carotenes (β-carotene and lycopene) and xanthophylls (astaxanthin, canthaxanthin, lutein, zeaxanthin, and capsanthin). Some biotechnological processes for carotenoids production were established some years ago, but new strains and technologies are being developed nowadays for carotenoids widely in demand. This chapter shows a revision of the main carotenoids from a commercial point of view.

Topics: beta Carotene; Biotechnology; Canthaxanthin; Carotenoids; Humans; Lutein; Lycopene; Xanthophylls; Zeaxanthins

Microalgae are the major photosynthesizers on earth and produce important pigments that include chlorophyll a, b and c, β-carotene, astaxanthin, xanthophylls, and phycobiliproteins. Presently, synthetic colorants are used in food, cosmetic, nutraceutical, and pharmaceutical industries. However, due to problems associated with the harmful effects of synthetic colorants, exploitation of microalgal pigments as a source of natural colors becomes an attractive option. There are various factors such as nutrient availability, salinity, pH, temperature, light wavelength, and light intensity that affect pigment production in microalgae. This paper reviews the availability and characteristics of microalgal pigments, factors affecting pigment production, and the application of pigments produced from microalgae. The potential of microalgal pigments as a source of natural colors is enormous as an alternative to synthetic coloring agents, which has limited applications due to regulatory practice for health reasons.

Topics: beta Carotene; Carotenoids; Chlorophyll; Coloring Agents; Hydrogen-Ion Concentration; Light; Microalgae; Phycobiliproteins; Pigments, Biological; Temperature; Xanthophylls

Cell, animal and human studies dealing with carotenoids and carotenoid derivatives as nutritional regulators of adipose tissue biology with implications for the etiology and management of obesity and obesity-related metabolic diseases are reviewed. Most studied carotenoids in this context are β-carotene, cryptoxanthin, astaxanthin and fucoxanthin, together with β-carotene-derived retinoids and some other apocarotenoids. Studies indicate an impact of these compounds on essential aspects of adipose tissue biology including the control of adipocyte differentiation (adipogenesis), adipocyte metabolism, oxidative stress and the production of adipose tissue-derived regulatory signals and inflammatory mediators. Specific carotenoids and carotenoid derivatives restrain adipogenesis and adipocyte hypertrophy while enhancing fat oxidation and energy dissipation in brown and white adipocytes, and counteract obesity in animal models. Intake, blood levels and adipocyte content of carotenoids are reduced in human obesity. Specifically designed human intervention studies in the field, though still sparse, indicate a beneficial effect of carotenoid supplementation in the accrual of abdominal adiposity. In summary, studies support a role of specific carotenoids and carotenoid derivatives in the prevention of excess adiposity, and suggest that carotenoid requirements may be dependent on body composition.

Topics: Adipocytes; Adipose Tissue; Animals; beta Carotene; Carotenoids; Cryptoxanthins; Humans; Obesity; Xanthophylls

Carotenoids are natural pigments with antioxidative functions that protect against oxidative stress. They are essential for humans and must be supplied through the diet. Carotenoids are the precursors for the visual pigment rhodopsin, and lutein and zeaxanthin must be accumulated in the yellow eye spot to protect the retina from excess light and ultraviolet damage. There is a global market for carotenoids as food colorants, animal feed, and nutraceuticals. Some carotenoids are chemically synthesized, whereas others are from natural sources. Microbial mass production systems of industrial interest for carotenoids are in use, and new ones are being developed by metabolic pathway engineering of bacteria, fungi, and plants. Several examples will be highlighted in this chapter.

Topics: Bacteria; beta Carotene; Biotechnology; Carotenoids; Coloring Agents; Dietary Supplements; Fungi; Industrial Microbiology; Light; Lutein; Metabolic Engineering; Oxidative Stress; Plants; Rhodopsin; Xanthophylls; Zeaxanthins

Cardiovascular disease related to atherosclerosis represents nowadays the largest cause of morbidity and mortality in developed countries. Due to inflammatory nature of atherosclerosis, several studies had been conducted in order to search for substances with anti-inflammatory activity on arterial walls, able to exert beneficial roles on health. Researches investigated the role of dietary carotenoids supplementation on cardiovascular disease, due to their free radicals scavenger properties and their skills in improving low-density lipoprotein cholesterol resistance to oxidation. Nevertheless, literature data are conflicting: although some studies found a positive relationship between carotenoids supplementation and cardiovascular risk reduction, others did not find any positive effects or even prooxidant actions. This paper aimed at defining the role of carotenoids supplementation on cardiovascular risk profile by reviewing literature data, paying attention to those carotenoids more present in our diet (β-carotene, α-carotene, β-cryptoxanthin, lycopene, lutein, zeaxanthin, and astaxanthin).

Topics: Animals; Anti-Inflammatory Agents; Antioxidants; Atherosclerosis; beta Carotene; Cardiovascular Diseases; Carotenoids; Cholesterol, LDL; Clinical Trials as Topic; Cryptoxanthins; Diet; Free Radical Scavengers; Humans; Lutein; Lycopene; Oxygen; Risk; Xanthophylls; Zeaxanthins

Astaxanthin is a red xanthophyll (oxygenated carotenoid) with large importance in the aquaculture, pharmaceutical, and food industries. The green alga Haematococcus pluvialis and the heterobasidiomycetous yeast Xanthophyllomyces dendrorhous are currently known as the main microorganisms useful for astaxanthin production at the industrial scale. The improvement of astaxanthin titer by microbial fermentation is a requirement to be competitive with the synthetic manufacture by chemical procedures, which at present is the major source in the market. In this review, we show how the isolation of new strains of X. dendrorhous from the environment, the selection of mutants by the classical methods of random mutation and screening, and the rational metabolic engineering, have provided improved strains with higher astaxanthin productivity. To reduce production costs and enhance competitiveness from an industrial point of view, low-cost raw materials from industrial and agricultural origin have been adopted to get the maximal astaxanthin productivity. Finally, fermentation parameters have been studied in depth, both at flask and fermenter scales, to get maximal astaxanthin titers of 4.7 mg/g dry cell matter (420 mg/l) when X. dendrorhous was fermented under continuous white light. The industrial scale-up of this biotechnological process will provide a cost-effective method, alternative to synthetic astaxanthin, for the commercial exploitation of the expensive astaxanthin (about $2,500 per kilogram of pure astaxanthin).

Topics: Basidiomycota; beta Carotene; Chlorophyta; Fermentation; Industrial Microbiology; Molecular Structure; Xanthophylls

Carotenoids are naturally occurring organic pigments that are believed to have therapeutic benefit in treating cardiovascular disease (CVD) because of their antioxidant properties. However, prospective randomized trials have failed to demonstrate a consistent benefit for the carotenoid beta-carotene in patients at risk for CVD. The basis for this apparent paradox is not well understood but may be attributed to the distinct antioxidant properties of various carotenoids resulting from their structure-dependent physicochemical interactions with biologic membranes. To test this hypothesis, we measured the effects of astaxanthin, zeaxanthin, lutein, beta-carotene, and lycopene on lipid peroxidation using model membranes enriched with polyunsaturated fatty acids. The correlative effects of these compounds on membrane structure were determined using small-angle x-ray diffraction approaches. The nonpolar carotenoids, lycopene and beta-carotene, disordered the membrane bilayer and stimulated membrane lipid peroxidation (>85% increase in lipid hydroperoxide levels), whereas astaxanthin (a polar carotenoid) preserved membrane structure and exhibited significant antioxidant activity (>40% decrease in lipid hydroperoxide levels). These results suggest that the antioxidant potential of carotenoids is dependent on their distinct membrane lipid interactions. This relation of structure and function may explain the differences in biologic activity reported for various carotenoids, with important therapeutic implications.

Topics: beta Carotene; Cardiovascular Diseases; Carotenoids; Endothelium, Vascular; Humans; Lipid Peroxidation; Lutein; Lycopene; Oxidative Stress; Xanthophylls; Zeaxanthins

Microalgae are a major natural source for a vast array of valuable compounds, including a diversity of pigments, for which these photosynthetic microorganisms represent an almost exclusive biological resource. Yellow, orange, and red carotenoids have an industrial use in food products and cosmetics as vitamin supplements and health food products and as feed additives for poultry, livestock, fish, and crustaceans. The growing worldwide market value of carotenoids is projected to reach over US$1,000 million by the end of the decade. The nutraceutical boom has also integrated carotenoids mainly on the claim of their proven antioxidant properties. Recently established benefits in human health open new uses for some carotenoids, especially lutein, an effective agent for the prevention and treatment of a variety of degenerative diseases. Consumers' demand for natural products favors development of pigments from biological sources, thus increasing opportunities for microalgae. The biotechnology of microalgae has gained considerable progress and relevance in recent decades, with carotenoid production representing one of its most successful domains. In this paper, we review the most relevant features of microalgal biotechnology related to the production of different carotenoids outdoors, with a main focus on beta-carotene from Dunaliella, astaxanthin from Haematococcus, and lutein from chlorophycean strains. We compare the current state of the corresponding production technologies, based on either open-pond systems or closed photobioreactors. The potential of scientific and technological advances for improvements in yield and reduction in production costs for carotenoids from microalgae is also discussed.

Topics: beta Carotene; Bioreactors; Carotenoids; Chlorophyta; Lutein; Xanthophylls

The low productivity of algal cultures in the production of high-value compounds is the most significant bottleneck for commercialization of this technology. Cultures in which cell mass is reused for continuous production are proposed as a solution to overcome this problem. Recently, a method was developed in which beta-carotene was harvested from the microalga Dunaliella salina grown in a two-phase bioreactor. This raises the question of whether this technique could also be used in the mass production of secondary metabolites. Understanding the mechanism of the milking process and its relationship to the product formation pathway should reveal whether other products can be milked from various species of microalgae.

Topics: beta Carotene; Bioreactors; Biotechnology; Cell Culture Techniques; Docosahexaenoic Acids; Eukaryota; Neurotoxins; Xanthophylls

Agricultural residues are abundant potential feedstocks for bioconversions to industrial fuels and chemicals. Every bushel of maize (approximately 25 kg) processed for sweeteners, oil, or ethanol generates nearly 7 kg of protein- and fiber-rich residues. Currently these materials are sold for very low returns as animal feed ingredients. Yeast-like fungi are promising biocatalysts for conversions of agricultural residues. Although corn fiber (pericarp) arabinoxylan is resistant to digestion by commercially available enzymes, a crude mixture of enzymes from the yeast-like fungus Aureobasidium partially saccharifies corn fiber without chemical pretreatment. Sugars derived from corn fiber can be converted to ethanol or other valuable products using a variety of naturally occurring or recombinant yeasts. Examples are presented of Pichia guilliermondii strains for the conversion of corn fiber hydrolysates to the alternative sweetener xylitol. Corn-based fuel ethanol production also generates enormous volumes of low-value stillage residues. These nutritionally rich materials are prospective substrates for numerous yeast fermentations. Strains of Aureobasidium and the red yeast Phaffia rhodozyma utilize stillage residues for production of the polysaccharide pullulan and the carotenoid astaxanthin, respectively.

Topics: beta Carotene; Ethanol; Fermentation; Glucans; Xanthophylls; Xylitol; Yeasts; Zea mays

The carotenoid pigment astaxanthin has important applications in the nutraceutical, cosmetics, food and feed industries. Haematococcus pluvialis is the richest source of natural astaxanthin and is now cultivated at industrial scale. Astaxanthin is a strong coloring agent and a potent antioxidant - its strong antioxidant activity points to its potential to target several health conditions. This article covers the antioxidant, UV-light protection, anti-inflammatory and other properties of astaxanthin and its possible role in many human health problems. The research reviewed supports the assumption that protecting body tissues from oxidative damage with daily ingestion of natural astaxanthin might be a practical and beneficial strategy in health management.

Topics: Adjuvants, Immunologic; Administration, Oral; Antioxidants; Arteriosclerosis; beta Carotene; Biological Availability; Blindness; Chlorophyta; Diet Therapy; Humans; Inflammation; Macular Degeneration; Neoplasms; Neurodegenerative Diseases; Nutritional Physiological Phenomena; Photosensitivity Disorders; Radiation-Sensitizing Agents; Species Specificity; Xanthophylls

The crystal structure of a beta-crustacyanin allows an analysis of the various proposals for the mechanism of the bathochromic shift from orange to purple-blue of astaxanthin in this lobster carotenoprotein. Structural and previous chemical and biophysical studies suggest that extension of conjugation by coplanarization of the beta-ionone rings with the polyene chain and polarization resulting from hydrogen bonding at the C(4) and C(4') keto groups may be mainly responsible for the bathochromic shift. Additional contributions may arise from medium effects and possibly from bowing of the polyene chain on binding. Previous biophysical data revealing a somewhat symmetrical polarization of astaxanthin in crustacyanin are thereby also accounted for. A puzzling feature remains unexplained: the bathochromic shifts, larger than that of astaxanthin, shown by some cyclopentenedione carotenoids in reconstituted carotenoproteins. This mini review enlarges on the original analysis and conclusions of Cianci et al. [(2002), Proc. Natl Acad. Sci. USA, 99, 9795-9800].

Topics: Animals; beta Carotene; Carotenoids; Carrier Proteins; Crystallography, X-Ray; Decapoda; Protein Conformation; Protein Structure, Tertiary; Proteins; Spectrum Analysis; Xanthophylls

Phaffia rhodozyma was isolated by Herman Phaff in the 1960s, during his pioneering studies of yeast ecology. Initially, the yeast was isolated from limited geographical regions, but isolates were subsequently obtained from Russia, Chile, Finland, and the United States. The biological diversity of the yeast is more extensive than originally envisioned by Phaff and his collaborators, and at least two species appear to exist, including the anamorph Phaffia rhodozyma and the teleomorph Xanthophyllomyces dendrorhous. The yeast has attracted considerable biotechnological interest because of its ability to synthesize the economically important carotenoid astaxanthin (3,3'-dihydroxy-beta, beta-carotene-4,4'-dione) as its major pigment. This property has stimulated research on the biology of the yeast as well as development of the yeast as an industrial microorganism for astaxanthin production by fermentation. Our laboratory has isolated several mutants of the yeast affected in carotenogenesis, giving colonies a vivid array of pigmentation. We have found that nutritional and environmental conditions regulate astaxanthin biosynthesis in the yeast, and have demonstrated that astaxanthin protects P. rhodozyma from damage by reactive oxygen species. We proposed in the 1970s that P. rhodozyma could serve as an economically important pigment source in animal diets including salmonids, lobsters, and the egg yolks of chickens and quail, in order to impart characteristic and desirable colors. Although P. rhodozyma/Xanthomyces dendrorhous has been studied by various researchers for nearly 30 years, it still attracts interest from yeast biologists and biotechnologists. There is a bright and colorful outlook for P. rhodozyma/X. dendrorhous from fundamental and applied research perspectives.

Topics: Basidiomycota; beta Carotene; Biotechnology; Carotenoids; History, 20th Century; Mycology; Xanthophylls

Biochemistry, biological crystallography, spectroscopy, solution X-ray scattering and microscopy have been applied to study the molecular basis of the colouration in lobster shell. This article presents a review of progress concentrating on recent results but set in the context of more than 50 years of work. The blue colouration of the carapace of the lobster Homarus gammarus is provided by a multimolecular carotenoprotein, alpha-crustacyanin. The complex is a 16-mer of five different subunits each binding the carotenoid, astaxanthin (AXT). A breakthrough in the structural studies came from the determination of the structure of beta-crustacyanin (protein subunits A1 with A3 with two shared bound astaxanthins). This was solved by molecular replacement using apocrustacyanin A1 as the search motif. A molecular movie has now been calculated by linear interpolation based on these two 'end-point' protein structures, i.e. apocrustacyanin A1 and A1 associated with the two astaxanthins in beta-crustacyanin, and is presented with this paper. This movie highlights the structural changes forced upon the carotenoid on complexation. In contrast, the protein-binding site remains relatively unchanged in the binding region, but there is a large conformational change occurring in a more remote surface-loop region. It is suggested here that this loop could be important in complexation of AXT and contributes to the spectral properties. Also presented here is the first observation of single-crystal diffraction of the full 'alpha-crustacyanin' complex comprising 16 protein subunits and 16 bound AXT molecules (i.e eight beta-crustacyanins) at 5 A resolution. Optimization of crystallization conditions is still necessary as these patterns show multiple crystallite character, however, 10 A resolution single-crystal diffraction has now been achieved. Provision of the new SRS MPW 10 and SRS MPW 14 beamline robotic systems will greatly assist in the surveying of the many alpha-crustacyanin crystallization trials that are being made. New solution X-ray scattering (SXS) measurements of beta- and alpha-crustacyanin are also presented. The beta-crustacyanin SXS data serve to show how the holo complex fits the SXS curve, whereas the apocrustacyanin A1 homodimer from the crystal data naturally does not. Reconstructions of alpha-crustacyanin were accomplished from its scattering-profile shape. The most plausible ultrastructure, based on a fourfold symmetry constraint, was found to be a stoo

Topics: Animals; beta Carotene; Binding Sites; Carrier Proteins; Crystallization; Crystallography, X-Ray; Microscopy, Electron; Models, Molecular; Nephropidae; Pigments, Biological; Protein Conformation; Proteins; Scattering, Radiation; Spectrophotometry; X-Rays; Xanthophylls

This review describes the different approaches that have been used to manipulate and improve carotenoid production in Xanthophyllomyces dendrorhous. The red yeast X. dendrorhous (formerly known as Phaffia rhodozyma) is one of the microbiological production systems for natural astaxanthin. Astaxanthin is applied in food and feed industry and can be used as a nutraceutical because of its strong antioxidant properties. However, the production levels of astaxanthin in wild-type isolates are rather low. To increase the astaxanthin content in X. dendrorhous, cultivation protocols have been optimized and astaxanthin-hyperproducing mutants have been obtained by screening of classically mutagenized X. dendrorhous strains. The knowledge about the regulation of carotenogenesis in X. dendrorhous is still limited in comparison to that in other carotenogenic fungi. The X. dendrorhous carotenogenic genes have been cloned and a X. dendrorhous transformation system has been developed. These tools allowed the directed genetic modification of the astaxanthin pathway in X. dendrorhous. The crtYB gene, encoding the bifunctional enzyme phytoene synthase/lycopene cyclase, was inactivated by insertion of a vector by single and double cross-over events, indicating that it is possible to generate specific carotenoid-biosynthetic mutants. Additionally, overexpression of crtYB resulted in the accumulation of beta-carotene and echinone, which indicates that the oxygenation reactions are rate-limiting in these recombinant strains. Furthermore, overexpression of the phytoene desaturase-encoding gene (crtI) showed an increase in monocyclic carotenoids such as torulene and HDCO (3-hydroxy-3',4'-didehydro-beta,-psi-carotene-4-one) and a decrease in bicyclic carotenoids such as echinone, beta-carotene and astaxanthin.

Topics: Basidiomycota; beta Carotene; Gene Expression Regulation, Fungal; Genetic Engineering; Recombinant Proteins; Xanthophylls

Antioxidants are substances capable of inhibiting oxidation. In chronic diseases, inflammatory response cells produce oxygen free radicals. Oxygen free radicals cause DNA damage, and this may lead to gene modifications that might be carcinogenic. Chronic Helicobacter pylori infection causes the production of DNA-damaging free radicals. In recent years, various groups have studied the effects of antioxidants, especially on H. pylori-associated gastric cancer. In most of the studies, it has been shown that H. pylori infection does affect the level of antioxidants measured in the gastric juice, but there are also controversial results. Recent experimental studies, both in vivo and in vitro, have shown that vitamin C and astaxanthin, a carotenoid, are not only free radical scavengers but also show antimicrobial activity against H. pylori. It has been shown that astaxanthin changes the immune response to H. pylori by shifting the Th1 response towards a Th2 T-cell response. Very few experimental studies support the epidemiologic studies, and further studies are needed to describe the effect and the mechanism of antioxidants in the H. pylori immune response.

Topics: Antioxidants; Ascorbic Acid; beta Carotene; DNA Damage; Helicobacter Infections; Helicobacter pylori; Humans; Risk Factors; Stomach Neoplasms; Th1 Cells; Th2 Cells; Xanthophylls

Dietary carotenoids react with a wide range of radicals such as CCl3O2*, RSO2*, NO2*, and various arylperoxyl radicals via electron transfer producing the radical cation of the carotenoid. Less strongly oxidizing radicals, such as alkylperoxyl radicals, can lead to hydrogen atom transfer generating the neutral carotene radical. Other processes can also arise such as adduct formation with sulphur-centered radicals. The oxidation potentials have been established, showing that, in Triton X-100 micelles, lycopene is the easiest carotenoid to oxidize to its radical cation and astaxanthin is the most difficult. The interaction of carotenoids and carotenoid radicals with other antioxidants is of importance with respect to anti- and possibly pro-oxidative reactions of carotenoids. In polar environments the vitamin E (alpha-tocopherol) radical cation is deprotonated (TOH*+ --> TO* + H+) and TO* does not react with carotenoids, whereas in nonpolar environments such as hexane, TOH*+ is converted to TOH by hydrocarbon carotenoids. However, the nature of the reaction between the tocopherol and various carotenoids shows a marked variation depending on the specific tocopherol homologue. The radical cations of the carotenoids all react with vitamin C so as to "repair" the carotenoid.

Topics: Adjuvants, Immunologic; Antioxidants; beta Carotene; Carotenoids; Cations; Detergents; Diet; Electrons; Free Radicals; Kinetics; Lycopene; Models, Chemical; Octoxynol; Protein Binding; Time Factors; Vitamin E; Xanthophylls

As a result of high production costs, commercial products from microalgae must command high prices. Astaxanthin produced by Haematococcus is a product that has become a commercial reality through novel and advanced technology. Cultivation methods have been developed to produce Haematococcus containing 1.5-3.0% astaxanthin by dry weight, with potential applications as a pigment source in aquaculture, poultry feeds and in the worldwide nutraceutical market.

Topics: Animal Feed; Animals; Aquaculture; beta Carotene; Biotechnology; Chlorophyta; Decapoda; Fishes; Food Coloring Agents; Poultry; Xanthophylls

Among the highly valued ketocarotenoids employed for food coloration, astaxanthin is probably the most important. This carotenoid may be produced biotechnologically by a number of microorganisms, and the most promising seems to be the freshwater flagellate Haematococcus pluvialis (Chlorophyceae), which accumulate astaxanthin in their aplanospores. Many physiological aspects of the transition of the flagellate into aplanospores have been described. Mixotrophic cultivation and suitable irradiance may result in fairly good yields (up to 40 mg/l; 43 mg/g cell dry weight) within a reasonable time, under laboratory conditions. In order to compete with synthetic astaxanthin, suitable scaling-up is required. However, large-scale production in open ponds has proved unsatisfactory because of severe contamination problems. A selective medium might overcome this difficulty. Further research for the development of suitable strains is thus warranted.

Topics: Basidiomycota; beta Carotene; Biotechnology; Carotenoids; Chlorophyta; Xanthophylls

The crt gene clusters responsible for the biosynthesis of carotenoids such as lycopene, beta-carotene and astaxanthin have been isolated from carotenogenic bacteria such as Erwinia species and the marine bacterium Agrobacterium aurantiacum. The functions of the individual genes have been identified. The first substrate of the enzymes encoded by the Erwinia crt clusters is farnesyl pyrophosphate which is not only the precursor for carotenoid biosynthesis but also sterols, dolichols and other numerous isoprenoid compounds. Escherichia coli does not naturally synthesize carotenoids, but by using the carotenogenic genes recombinant strains accumulating lycopene, beta-carotene and astaxanthin have been produced. Other non-carotenogenic bacteria such as Zymomonas mobilis have also been engineered to produce beta-carotene by the introduction of the corresponding crt genes. A gene capable of enhancing carotenoid levels in E. coli has also been isolated from cDNA libraries of the yeast Phaffia rhodozyma and the green alga Haematococcus pluvialis. This gene has been found to encode an isopentenyl pyrophophate isomerase. It has further been shown that the edible yeasts Candida utilis as well as Saccharomyces cerevisiae, which possess no carotenoid biosynthetic pathway, acquire the ability to produce carotenoids, when the carotenogenic genes are expressed under the control of yeast-derived promoters and terminators. It has been observed in the yeasts S. cerevisiae and C. utilis carrying the lycopene biosynthesis genes that ergosterol content is decreased by 10 and 35%, respectively. It is therefore likely that the carbon flux for the ergosterol biosynthesis has been partially directed from farnesyl pyrophosphate to a new pathway for the lycopene biosynthesis. Further, the expression of a truncated gene which codes for the catalytic domain of the endogenous 3-hydroxy-3-methylglutaryl coenzyme. A reductase, has been found to be effective for enhancing carotenoid levels in the yeast C. utilis.

Topics: beta Carotene; Carotenoids; Escherichia coli; Genes, Bacterial; Genes, Fungal; Genetic Engineering; Lycopene; Molecular Structure; Transformation, Genetic; Xanthophylls

Topics: Animals; beta Carotene; Carotenoids; Free Radical Scavengers; Lutein; Molecular Structure; Reactive Oxygen Species; Vitamin E; Xanthophylls

Topics: Absorption; Animal Nutritional Physiological Phenomena; Animals; beta Carotene; Birds; Carotenoids; Chemical Phenomena; Chemistry; Chick Embryo; Chickens; Fishes; Goldfish; Hydrocarbons; Insecta; Lutein; Neoplasms, Experimental; Oxidation-Reduction; Photosensitivity Disorders; Pigmentation; Pigments, Biological; Retina; Salmon; Skin; Trout; Vitamin A; Xanthophylls

Topics: Absorption; Animals; beta Carotene; Carotenoids; Cryptoxanthins; Food Analysis; Humans; Lutein; Lycopene; Models, Biological; Nutritional Requirements; Structure-Activity Relationship; Vitamin A; Xanthophylls

Carotenoids have been implicated in the regulation of adipocyte metabolism.. To compare the effects of mixed-carotenoid supplementation (MCS) versus placebo on adipokines and the accrual of abdominal adiposity in children with obesity.. Randomized (1:1), double-blind, placebo-controlled intervention trial to evaluate the effects of MCS over 6 months in a subspecialty clinic.. Twenty (6 male and 14 female) children with simple obesity [body mass index (BMI) > 90%], a mean age (± standard deviation) of 10.5 ± 0.4 years, and Tanner stage I to V were enrolled; 17 participants completed the trial.. MCS (which contains β-carotene, α-carotene, lutein, zeaxanthin, lycopene, astaxanthin, and γ-tocopherol) or placebo was administered daily.. Primary outcomes were change in β-carotene, abdominal fat accrual (according to magnetic resonance imaging), and BMI z-score; secondary outcomes were adipokines and markers of insulin resistance.. Cross-sectional analysis of β-carotene showed inverse correlation with BMI z-score, waist-to-height ratio, visceral adipose tissue, and subcutaneous adipose tissue (SAT) at baseline. MCS increased β-carotene, total adiponectin, and high-molecular-weight adiponectin compared with placebo. MCS led to a greater reduction in BMI z-score, waist-to-height ratio, and SAT compared with placebo. The percentage change in β-carotene directly correlated with the percentage change in SAT.. The decrease in BMI z-score, waist-to-height ratio, and SAT and the concomitant increase in the concentration of β-carotene and high-molecular-weight adiponectin by MCS suggest the putative beneficial role of MCS in children with obesity.

Topics: Abdominal Fat; Adipokines; Adiponectin; beta Carotene; Carotenoids; Child; Double-Blind Method; Female; gamma-Tocopherol; Humans; Intra-Abdominal Fat; Lutein; Lycopene; Magnetic Resonance Imaging; Male; Obesity, Abdominal; Pediatric Obesity; Pilot Projects; Subcutaneous Fat; Waist-Height Ratio; Xanthophylls; Zeaxanthins

Topics: Administration, Oral; Antioxidants; Ascorbic Acid; beta Carotene; Carotenoids; Double-Blind Method; Drug Combinations; Erythema; Female; Humans; Lutein; Lycopene; Male; Radiation Dosage; Time Factors; Ultraviolet Rays; Vitamin E; Xanthophylls

A randomized, double-blind human trial was conducted to assess the effect on the plasma carotenoid concentration of 4- or 12-week astaxanthin supplementation (1 or 3 mg/d) of 20 Japanese middle-aged and senior subjects. The plasma carotenoid concentration was significantly higher after the astaxanthin supplementation than that before in both the 1 mg/d (10 subjects) and 3 mg/d (10 subjects) groups.

Topics: Administration, Oral; Aged; Antioxidants; beta Carotene; Biological Availability; Carotenoids; Chromatography, High Pressure Liquid; Dietary Supplements; Double-Blind Method; Drug Administration Schedule; Female; Humans; Japan; Lutein; Lycopene; Male; Middle Aged; Oxidative Stress; Tandem Mass Spectrometry; Xanthophylls

To evaluate the treatment of male infertility with a strong natural antioxidant, in addition to conventional treatment.. Using a double blind, randomized trial design, 30 men with infertility of > or =2 months and female partners with no demonstrable cause of infertility received conventional treatment according to the guidelines of the World Health Organization (WHO), and either a strong antioxidant Astaxanthin 16 mg/day (AstaCarox, AstaReal AB, Gustavsberg, Sweden) or placebo for 3 months. The effects of treatment on semen parameters, reactive oxygen species (ROS), zona-free hamster oocyte test, serum hormones including testosterone, luteinizing hormone (LH), follicle stimulating hormone (FSH) and Inhibin B, and spontaneous or intrauterine insemination (IUI)-induced pregnancies were evaluated.. ROS and Inhibin B decreased significantly and sperm linear velocity increased in the Astaxanthin group (n = 11), but not in the placebo group (n = 19). The results of the zona-free hamster oocyte test tended to improve in the Astaxanthin group in contrast with the placebo group, though not reaching statistical significance. The total and per cycle pregnancy rates among the placebo cases (10.5 % and 3.6 %) were lower compared with 54.5 % and 23.1 % respectively in the Astaxanthin group (P = 0.028; P = 0.036).. Although the present study suggests a positive effect of Astaxanthin on sperm parameters and fertility, the results need to be confirmed in a larger trial before recommending Astaxanthin for the complementary treatment of infertile men.

Topics: Adult; Antioxidants; beta Carotene; Double-Blind Method; Female; Humans; Infertility, Male; Male; Placebos; Pregnancy; Pregnancy Rate; Prospective Studies; Xanthophylls

This investigation was designed to determine the effects of astaxanthin on markers of skeletal muscle injury. Twenty resistance trained men (mean +/- standard error of the mean : age , 25.1 +/- 1.6 y; height, 1.79 +/- 0.02 m; weight, 86.8 +/- 4.4 kg) were assigned to either a placebo (1732 mg safflower oil, n =10) or astaxanthin (BioAstin; 1732 mg safflower oil; haematococcus algae extract [contains 4 mg astaxanthin and 480 mg lutein]n = 10). Subjects consumed their assigned treatment for 3 wk prior to eccentric exercise (10 sets of 10 repetitions at 85% of one repetition maximum) and through 96 h post-exercise. Muscle soreness, creatine kinase (CK), and muscle performance was measured before and through 96 h post-exercise. A similar response was observed for both treatment groups for all dependent variables, indicating that in resistance trained men, astaxanthin supplementation does not favorably affect indirect markers of skeletal muscle injury following eccentric loading.

Topics: Adjuvants, Immunologic; Adult; beta Carotene; Creatine Kinase; Dietary Supplements; Double-Blind Method; Exercise; Humans; Male; Muscle Fatigue; Muscle, Skeletal; Pain Measurement; Weight Lifting; Xanthophylls

A growing body of scientific literature indicates that astaxanthin is a more powerful antioxidant than other carotenoids and vitamin E and may confer numerous health benefits. The purpose of this investigation was to conduct a human safety study with a Haematococcus pluvialis algal extract with high levels of astaxanthin. Thirty-five healthy adults age 35-69 years were enrolled in a randomized, double-blind, placebo-controlled trial of 8 weeks' duration. All participants took three gelcaps per day, one at each meal. Nineteen participants received gelcaps with an algal extract in safflower oil, containing 2 mg of astaxanthin each (treatment); 16 participants received gelcaps containing safflower oil only (placebo). Blood pressure and blood chemistry tests, including a comprehensive metabolic panel and cell blood count, were conducted at the beginning of the trial and after 4 and 8 weeks of supplementation. No significant differences were detected between the treatment and the placebo groups after 8 weeks of supplementation with the algal extract in the parameters analyzed, except for serum calcium, total protein, and eosinophils (P <.01). Although the differences in these three parameters were statistically significant, they were very small and are of no clinical importance. These results reveal that 6 mg of astaxanthin per day from a H. pluvialis algal extract can be safely consumed by healthy adults.

Topics: Adult; Aged; beta Carotene; Blood Pressure; Blood Proteins; Calcium; Eosinophils; Erythrocyte Count; Eukaryota; Female; Humans; Leukocyte Count; Male; Middle Aged; Placebos; Xanthophylls

Astaxanthin and β-carotene are the most prominent carotenoids extensively used in pharmaceutics. Here, we present a halotolerant bacterium from Lake Wadi El-Natrun capable of producing astaxanthin and β-carotene analyzed by HPLC, ESI-MS, and infrared spectroscopy. The phenotypic and phylogenetic analyses classified the isolate as a novel strain of the genus Planococcus, for which the name Planococcus sp. Eg-Natrun is proposed. Carotenoid biosynthesis can exceptionally occur in a light-inducible or constitutive manner. The maximum carotenoid yields were 610 ± 13 µg/g (~ 38% β-carotene and ~ 21% astaxanthin) in a minimal medium with acetate and 1024 ± 53 µg/g dry cells in a rich marine medium. The carotenogenesis incentives (e.g., acetate) and disincentives (e.g., methomyl) were discussed. Moreover, we successfully isolated the CrtE gene, one of the astaxanthin biosynthesis genes, from the unknown genome using a consensus-based degenerate PCR approach. To our knowledge, this is the first report elucidating astaxanthin and β-carotene in the genus Planococcus.

Topics: beta Carotene; Carotenoids; Phylogeny; Xanthophylls

Aurantiochytrium limacinum is a heterotrophic eukaryotic microorganism that can accumulate high levels of commercial products such as astaxanthin and docosahexaenoic acid. Due to its rapid growth and relatively simple extraction method, A. limacinum is considered a promising astaxanthin resource to replace the conventional microalgal production. However, the astaxanthin biosynthetic process in A. limacinum remains incompletely understood, especially in those catalysed by β-carotene hydroxylase (CrtZ) and ketolase. In this study, we overexpressed a crtZ candidate gene to increase astaxanthin production and expand our understanding of the conversion from beta-carotene to astaxanthin. The resultant transformant AlcrtZ#10 cultivated for 5 days showed a significant increase in astaxanthin production per culture (2.8-fold) and per cell (4.5-fold) compared with that of the wild-type strain. Strikingly, longer light exposure increased astaxanthin production and decreased the beta-carotene content in the wild-type strain, suggesting that light exposure duration is important for astaxanthin production in A. limacinum. Among several predicted intermediates, furthermore, the cantaxanthin produced from β-carotene by ketolase activity were enhanced in the transformant AlcrtZ#10. Although the further investigation is needed, this result suggested that the main route of astaxanthin was via cantaxanthin. Thus, our findings will be valuable not only for its application, but also for understanding the astaxanthin biosynthetic process in A. limacinum.

Topics: beta Carotene; Mixed Function Oxygenases; Oxygenases

The red velvet mite, Balaustium murorum (Hermann), is a pollenophagous free-living mite with a flashy red body. This mite occurs in early spring and lives on sunny surfaces of human-made structures, such as concrete. Hence, it is inevitably exposed to a harsh environment due to solar ultraviolet-B (UV-B) radiation and radiant heat, which cause oxidative stress via the production of reactive oxygen species. The spider mite Panonychus citri that resides on upper leaf surfaces accumulates synthesized keto-carotenoids to protect against oxidative stress. Therefore, we evaluated carotenoid composition in the red pigment of B. murorum. To identify major carotenoids, we performed a high-performance liquid chromatography analysis of intact and de-esterified pigments of B. murorum females. The flashy red pigments of B. murorum consisted of the highly abundant keto-carotenoids astaxanthin and 3-hydroxyechinenone (60 and 38% of major carotenoids, respectively), and a small amount of β-carotene (2%). Although P. citri is an astaxanthin-rich species, the astaxanthin concentration (per protein) in B. murorum is 127-fold that in P. citri. Due to their high antioxidant activities, those keto-carotenoids probably contribute to the survival of B. murorum in the harsh environment caused by solar UV-B radiation and radiant heat in inorganic habitats.

Topics: Animals; beta Carotene; Carotenoids; Female; Humans; Xanthophylls

Lycopene β-cyclase (EC 5.5.1.19) is one of the key enzymes in the biosynthesis of β-carotene and derived carotenoids. It catalyzes isomerase reactions to form β-carotene from lycopene by β-cyclization of both of its ψ-ends. Lycopene β-cyclases are widespread in nature. We systematically analyzed the phylogeny of lycopene β-cyclases from all kingdoms of life and predicted their transmembrane structures. To this end, a collection of previously characterized lycopene β-cyclase polypeptide sequences served as bait sequences to identify their closest homologues in a range of bacteria, archaea, fungi, algae, and plant species. Furthermore, a DeepTMHMM scan was applied to search for the presence of transmembrane domains. A phylogenetic tree suggests at least five distinct clades, and the DeepTMHMM scan revealed that lycopene β-cyclases are a group of structurally different proteins: membrane-bound and cytosolic enzymes. Representative lycopene β-cyclases were screened in the lycopene-overproducing

Topics: beta Carotene; Corynebacterium glutamicum; Intramolecular Lyases; Lycopene; Phylogeny

This study aimed to produce carotenoids by Phaffia rhodozyma in a stirred-tank bioreactor under the influence of magnetic fields (MF) and to evaluate a sustainable approach to recover them from the yeast biomass. MF application proved to be effective in increasing 8.6 and 22.9 % of β-carotene and astaxanthin production, respectively. Regarding solid-liquid extraction (SLE), the ability of aqueous and ethanolic solutions of protic ionic liquids (PILs) was determined. β-carotene and astaxanthin recovery yields increased with the anion alkyl chain length hydrophobicity. [Pro][Oct]:EtOH (50 % v v

Topics: Basidiomycota; beta Carotene; Bioreactors; Carotenoids; Ethanol; Saccharomyces cerevisiae

Dunaliella salina can accumulate a large amount of β-carotene which is generally considered to be its terminal product of carotenoid metabolism. In this study, it was proved that D. salina has the ketolase (DsBKT) of catalyzing the synthesis of astaxanthin, the downstream products of β-carotene. Therefore, the reason why D. salina does not synthesize astaxanthin is the purpose of this study. The enzymatic activity of DsBKT was detected by functional complementation assays in Escherichia coli, results showed that DsBKT had efficient ketolase activity toward β-carotene and zeaxanthin to produce astaxanthin, indicating that there were complete astaxanthin-producing genes in Dunaliella. Unlike the induced expression of Lycopene cyclase (catalyzing β-carotene synthesis) under salt stress, the expression of DsBKT was very low under both normal and stress conditions, which may be the main reason why D. salina cannot accumulate astaxanthin. On the contrary, with the astaxanthin-rich Haematococcus pluvialis as a control, its BKT gene was significantly upregulated under salt stress. Further study showed that DsBKT promoter had strong promoter ability and could stably drive the expression of ble-egfp in D. salina. Obviously, DsBKT promoter is not the reason of DsBKT not being expressed which may be caused by Noncoding RNA.

Topics: beta Carotene; Chlorophyta; Escherichia coli; Oxygenases; Salt Stress; Xanthophylls

There is a growing demand in the development of environmentally friendly technologies, based on the use of more biocompatible solvents for the recovery of natural bioactive compounds. In this work, the red yeast Phaffia rhodozyma biomass was used as a source of carotenoids to develop an integrative and efficient platform that promotes the recovery of astaxanthin and β-carotene using bio-based solvents (BioSs). The extraction aptitude of pure BioSs was evaluated and compared with the conventional organic method. At this point, the influence of the BioSs molecular structures involved in the extraction procedures were also investigated. Overall, envisaging the industrial application of the process, an integrative platform was proposed for the recovery of astaxanthin/β-carotene from P. rhodozyma biomass and the recycle of the BioSs. The life cycle assessment of the proposed technology using EtOH was evaluated, validating the sustainability of BioSs in the process with environmental impact reduction of 3-12%.

Topics: Basidiomycota; beta Carotene; Biomass; Solvents; Xanthophylls

The aim of this work was to uncover the astaxanthin biosynthesis mechanism in Microcystis aeruginosa under optimum light quality, and promote astaxanthin production using this alga. Among purple, blue and red light, only purple light promoted M. aeruginosa cell growth compared with white light, due to up-regulating expression of the genes related with DNA replication. An increase was detected in the photosynthetic rate under purple light, which should be caused by the raised carotenoid content and up-regulation of the genes associated with light reaction and carbon fixation. Compared with white light, purple light increased the levels of β-carotene, zeaxanthin and astaxanthin by up-regulating expression of the genes related with methylerythritol-4-phosphate pathway (MEP) and astaxanthin biosynthesis. For red and blue light, they did not impact or declined the content of astaxanthin and its precursors. Therefore, purple light promoted M. aeruginosa cell growth and astaxanthin production by up-regulating related gene expression.

Topics: beta Carotene; Light; Microcystis; Xanthophylls

Carotenoids are important photosynthetic pigments with many physiological functions, nutritional properties and high commercial value. β-carotene hydroxylase is one of the key enzymes in the carotenoid synthesis pathway of Chlamydomonas reinhardtii for the conversion of β-carotene to astaxanthin. The vector p64DZ containing the β-carotene hydroxylase gene crtZ from Haematococcus pluvialis was transformed into C. reinhardtii CC-503. The transformants were selected by alternate culture in solid-liquid medium containing spectinomycin (100 µg mL

Topics: beta Carotene; Chlamydomonas reinhardtii; Mixed Function Oxygenases; Xanthophylls

Astaxanthin is a kind of ketone carotenoid belonging to tetraterpenoids with an excellent antioxidant activity and it is widely used in nutrition and health-care industries. This study aimed to explore the effect of different abiotic stresses on carotenoid production in Schizochytrium sp. Firstly, the characteristics of carotenoid accumulation were studied in Schizochytrium sp. by monitoring the change of carotenoid yields and gene expressions. Then, different abiotic stresses were systematically studied to regulate the carotenoid accumulation. Results showed that low temperature could advance the astaxanthin accumulation, while ferric ion could stimulate the conversion from carotene to astaxanthin. The glucose and monosodium glutamate ratio of 100:5 was helpful for the accumulation of β-carotene. In addition, micro-oxygen supply conditions could increase the yield of β-carotene and astaxanthin by 25.47% and 14.92%, respectively. This study provided the potential regulation strategies for carotenoid production which might be used in different carotenoid-producing strains.

Topics: beta Carotene; Carotenoids; Stramenopiles; Stress, Physiological; Xanthophylls

Topics: Antioxidants; beta Carotene; Carotenoids; Chlorophyceae; Chlorophyta; Herbicides; Hydrogen Peroxide; Microalgae; Oxidants; Plant Extracts; Plant Growth Regulators; Sodium Chloride; Xanthophylls

The bifunctional enzyme β-carotene hydroxylase (CrtZ) catalyzes the hydroxylation of carotenoid β-ionone rings at the 3, 3' position regardless of the presence of keto group at 4, 4' position, which is an important step in the synthesis of astaxanthin. The level and substrate preference of CrtZ may have great effect on the amount of astaxanthin and the accumulation of intermediates.. In this study, the substrate preference of PCcrtZ from Paracoccus sp. PC1 and PAcrtZ from Pantoea Agglomerans were certified and were combined utilization for increase astaxanthin production. Firstly, PCcrtZ from Paracoccus sp. PC1 and PAcrtZ from P. Agglomerans were expressed in platform strains CAR032 (β-carotene producing strain) and Can004 (canthaxanthin producing strain) separately to identify their substrate preference for carotenoids with keto groups at 4,4' position or not. The results showed that PCcrtZ led to a lower zeaxanthin yield in CAR032 compared to that of PAcrtZ. On the contrary, higher astaxanthin production was obtained in Can004 by PCcrtZ than that of PAcrtZ. This demonstrated that PCCrtZ has higher canthaxanthin to astaxanthin conversion ability than PACrtZ, while PACrtZ prefer using β-carotene as substrate. Finally, Ast010, which has two copies of PAcrtZ and one copy of PCcrtZ produced 1.82 g/L of astaxanthin after 70 h of fed-batch fermentation.. Combined utilization of crtZ genes, which have β-carotene and canthaxanthin substrate preference respectively, can greatly enhance the production of astaxanthin and increase the ratio of astaxanthin among total carotenoids.

Topics: beta Carotene; Canthaxanthin; Carotenoids; Escherichia coli; Oxygenases; Paracoccus; Xanthophylls

Astaxanthin is a type of carotenoid widely used as powerful antioxidant and colourant in aquaculture and the poultry industry. Production of astaxanthin by yeast Xanthophyllomyces dendrorhous has attracted increasing attention due to high cell density and low requirements of water and land compared to photoautotrophic algae. Currently, the regulatory mechanisms of astaxanthin synthesis in X. dendrorhous remain obscure. In this study, we obtained a yellow X. dendrorhous mutant by Atmospheric and Room Temperature Plasma (ARTP) mutagenesis and sequenced its genome. We then identified a putative GATA transcription factor, white collar 2 (XdWC2), from the comparative genome data and verified that disruption of the XdWC2 gene resulted in a similar carotenoid profile to that of the ARTP mutant. Furthermore, transcriptomic analysis and yeast one-hybrid (Y1H) assay showed that XdWC2 regulated the expression of phytoene desaturase gene CrtI and astaxanthin synthase gene CrtS. The yeast two-hybrid (Y2H) assay demonstrated that XdWC2 interacted with white collar 1 (XdWC1) forming a heterodimer WC complex (WCC) to regulate the expression of CrtI and CrtS. Increase of the transcriptional levels of XdWC2 or CrtS in the wild-type strain did not largely modify the carotenoid profile, indicating translational and/or post-translational regulations involved in the biosynthesis of astaxanthin. Overexpression of CrtI in both the wild-type strain and the XdWC2-disrupted strain apparently improved the production of monocyclic carotenoid 3-hydroxy-3', 4'-didehydro-β, ψ-carotene-4-one (HDCO) rather than β-carotene and astaxanthin. The regulation of carotenoid biosynthesis by XdWC2 presented here provides the foundation for further understanding the global regulation of astaxanthin biosynthesis and guides the construction of astaxanthin over-producing strains.

Topics: Antioxidants; Basidiomycota; beta Carotene; Carotenoids; Fungal Proteins; GATA Transcription Factors; Saccharomyces cerevisiae; Water; Xanthophylls

Recent studies indicated a strong relationship between carotenoids and gut microflora. However, their structure-activity relationship remains unclear. This study evaluated the interaction between four typical carotenoids (β-carotene, lutein, lycopene, and astaxanthin) and gut microflora using an

Topics: beta Carotene; Carotenoids; Fermentation; Gastrointestinal Microbiome; Humans; Lutein; Lycopene; Xanthophylls; Zeaxanthins

The antioxidant activity of food compounds is one of the properties generating the most interest, due to its health benefits and correlation with the prevention of chronic disease. This activity is usually measured using in vitro assays, which cannot predict in vivo effects or mechanisms of action. The objective of this study was to evaluate the in vivo protective effects of six phenolic compounds (naringenin, apigenin, rutin, oleuropein, chlorogenic acid, and curcumin) and three carotenoids (lycopene B, β-carotene, and astaxanthin) naturally present in foods using a zebrafish embryo model. The zebrafish embryo was pretreated with each of the nine antioxidant compounds and then exposed to tert-butyl hydroperoxide (tBOOH), a known inducer of oxidative stress in zebrafish. Significant differences were determined by comparing the concentration-response of the tBOOH induced lethality and dysmorphogenesis against the pretreated embryos with the antioxidant compounds. A protective effect of each compound, except β-carotene, against oxidative-stress-induced lethality was found. Furthermore, apigenin, rutin, and curcumin also showed protective effects against dysmorphogenesis. On the other hand, β-carotene exhibited increased lethality and dysmorphogenesis compared to the tBOOH treatment alone.

Topics: Animals; Antioxidants; Apigenin; beta Carotene; Biological Factors; Carotenoids; Curcumin; Disease Models, Animal; Dose-Response Relationship, Drug; Embryo, Nonmammalian; Embryonic Development; Flavanones; Lycopene; Oxidative Stress; Polyphenols; tert-Butylhydroperoxide; Xanthophylls; Zebrafish

Inhibition of HMG-CoA (3-hydroxy-3-methylglutaryl coenzyme A) reductase, the rate rate-determining enzyme for the biogenesis of cholesterol is known to show antineoplastic effects. Therefore, this study investigates the in-silico HMG-CoA reductase (HMGCR)-inhibitory and in-vivo anti-lipidaemic/anticancer effects of carotenoids from Spondias mombin.. Carotenoids from S. mombin leaves were characterized with the aid of liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS). The characterized phytochemicals were obtained from PubChem. They were docked into the orthosteric site of human HMGCR (Protein Data Bank code 1HW8) using AutoDock 4.0 suites. DMBA (7,12-dimethylbenz[a]anthracene) model of breast cancer was treated with the carotenoids extract from S. mombin (100 mg/kg and 200 mg/kg doses) to assess its anti-lipidaemic cum anticancer effects.. Carotenoids from S. mombin; beta-carotene-15,15'-epoxide, astaxanthin and 7,7',8,8'-tetrahydro-β-β-carotene demonstrate HMGCR inhibition. They form hydrophobic interactions with key residues within the catalytic domain of HMGCR. The carotenoids extract exhibits anti-lipidaemic/anticancer effects, lowering serum triglyceride, LDL and cholesterol concentration. It increases HDL concentration and downregulates the expression of HMGR, AFP, CEACAM-3, BRCA-1 and HIF-1 mRNAs.. Carotenoids from S. mombin demonstrate HMG-CoA reductase (HMGCR) inhibition, anti-lipidaemic, and anticancer effects. The inhibition of HMGCR by the carotenoids extract further poses it as a potential anti-hypercholesterolaemia compounds.

Topics: Acyl Coenzyme A; Anacardiaceae; Animals; Anticholesteremic Agents; Antineoplastic Agents, Phytogenic; beta Carotene; Breast; Breast Neoplasms; Carotenoids; Down-Regulation; Female; Humans; Hydroxymethylglutaryl CoA Reductases; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Hypercholesterolemia; Hyperlipidemias; Hypolipidemic Agents; Lipids; Molecular Docking Simulation; Phytotherapy; Plant Extracts; Rats, Wistar; Xanthophylls

The antioxidant factors, astaxanthin, β-carotene, and resveratrol, have a potential effect on protein synthesis in skeletal muscle and a combined intake may have a greater cumulative effect than individual intake. The aim of this study was to investigate the combined effects on skeletal muscle mass and protein metabolic signaling during the hypertrophic process from atrophy in mice.. Male ICR mice were divided into five dietary groups consisting of seven animals each: normal, astaxanthin, β-carotene, resveratrol, and all three antioxidants. Equal concentrations (0.06% [w/w]) of the respective antioxidants were included in the diet of each group. In the mixed group, three antioxidants were added in equal proportion. One leg of each mouse was casted for 3 wk to induce muscle atrophy. After removal of the cast, the mice were fed each diet for 2 wk. The muscle tissues were collected, weighed, and examined for protein metabolism signaling and oxidative damage.. The weight of the soleus muscle was increased in the astaxanthin, β-carotene, and resveratrol groups to a greater extent than in the normal group; this was accelerated by intake of the mixed antioxidants (P = 0.007). Phosphorylation levels of mammalian target of rapamycin and p70 S6 K in the muscle were higher in the mixed antioxidant group than in the normal group (P = 0.025; P = 0.020). The carbonylated protein concentration was lower in the mixed antioxidant group than in the normal group (P = 0.021).. These results suggested that a combination of astaxanthin, β-carotene, and resveratrol, even in small amounts, promoted protein synthesis during the muscle hypertrophic process following atrophy.

Topics: Animals; Antioxidants; beta Carotene; Diet; Hypertrophy; Male; Mice; Mice, Inbred ICR; Muscle, Skeletal; Muscular Atrophy; Oxidative Stress; Protein Biosynthesis; Resveratrol; Xanthophylls

Carotenogenic microalgae are unicellular photosynthetic organisms with the ability to accumulate carotenoids. Carotenoid accumulation is a protective reaction against environmental stress factors, such as bright light and extreme temperatures. It makes the survival of these microorganisms under harsh environmental conditions possible. The diversity of carotenogenic microalgae has been described in detail for Central Europe and North America, as well as for tropical and subtropical latitudes with relatively favorable environments. However, data about these microorganisms in polar and subpolar latitudes are scarce and restricted to few reports. We isolated several strains of carotenogenic microalgae from the coastal zone of the White Sea, where they were abundant. The obtained microalgae related to four species of Chlorophytes: Haematococcus lacustris, H. rubicundus, Coelastrella aeroterrestrica and Bracteacoccus aggregatus. The last three species have been reported for polar latitudes for the first time. Most likely, carotenogenic algae in the White Sea coast are abundant due to their high physiological and metabolic plasticity, which is essential for surviving under adverse conditions of the northern regions. Pigment composition of the strains is provided. Their predominant carotenoids were astaxanthin and β-carotene. Further, the obtained strains may be considered as potential producers of natural pigments for biotechnology.

Topics: Arctic Regions; beta Carotene; Biodiversity; Carotenoids; Chlorophyceae; Cold Climate; Microalgae; Oceans and Seas; Photosynthesis; Xanthophylls

Effects of natural catalysts, isothiocyanates and polysulfides, on

Topics: beta Carotene; Carotenoids; Catalysis; Isomerism; Isothiocyanates; Lycopene; Sulfides; Xanthophylls

Topics: Animals; beta Carotene; Brachyura; Diet; Gene Expression Profiling; Gene Expression Regulation; Hemolymph; Hepatopancreas; Xanthophylls

Carotenoids are essential in oxygenic photosynthesis: they stabilize the pigment-protein complexes, are active in harvesting sunlight and in photoprotection. In plants, they are present as carotenes and their oxygenated derivatives, xanthophylls. While mutant plants lacking xanthophylls are capable of photoautotrophic growth, no plants without carotenes in their photosystems have been reported so far, which has led to the common opinion that carotenes are essential for photosynthesis. Here, we report the first plant that grows photoautotrophically in the absence of carotenes: a tobacco plant containing only the xanthophyll astaxanthin. Surprisingly, both photosystems are fully functional despite their carotenoid-binding sites being occupied by astaxanthin instead of β-carotene or remaining empty (i.e. are not occupied by carotenoids). These plants display non-photochemical quenching, despite the absence of both zeaxanthin and lutein and show that tobacco can regulate the ratio between the two photosystems in a very large dynamic range to optimize electron transport.. Most life on Earth depends on photosynthesis, the process used by plants and many other organisms to store energy from sunlight and produce oxygen. The first steps of photosynthesis, the capture and conversion of sunlight into chemical energy, happen in large assemblies of proteins containing many pigment molecules called photosystems. In plants, the pigments involved in photosynthesis are green chlorophylls and carotenoids. In addition to harvesting light, carotenoids have an important role in preventing damage caused by overexposure to sunlight There are over one thousand different carotenoids in living beings, but only one, β-carotene, is present in every organism that performs the type of photosynthesis in which oxygen is released, and is thought to be essential for the process. However, this could never be proved because it is impossible to remove β-carotene from cells using typical genetic approaches without affecting all other carotenoids. Xu et al. used genetic engineering to create tobacco plants that produced a pigment called astaxanthin in place of β-carotene. Astaxanthin is a carotenoid from salmon and shrimp, not normally found in plants. These plants are the first living things known to perform photosynthesis without β-carotene and demonstrate that this pigment is not essential for photosynthesis as long as other carotenoids are present. Xu et al. also show that the photosystems can adapt to using different carotenoids, and can even operate with a reduced number of them. Xu et al’s findings show the high flexibility of photosynthesis in plants, which are able to incorporate non-native elements to the process. These results are also important in the context of increasing the photosynthetic efficiency, and thus the productivity of crops, since they show that a radical redesign of the photosynthetic machinery is feasible.

Topics: beta Carotene; Nicotiana; Photosynthesis; Plants, Genetically Modified; Xanthophylls

Carotenoid pigments are valuable components of the human diet. A notable example is β-carotene, or provitamin A, which is converted into the derivatives astaxanthin and capsanthin, via the common intermediate zeaxanthin. To generate rice varieties producing diverse carotenoids beyond β-carotene, we specifically used a Capsicum β-carotene hydroxylase gene, B (CaBch) and a codon optimized version of the same gene, stB (stBch) to increase zeaxanthin synthesis. We also used a recombinant BAK gene (CaBch-2A-HpBkt), consisting of the CaBch sequence and a Haematococcus β-carotene ketolase gene (HpBkt) linked by a bicistronic 2 A sequence, as well as a codon optimized recombinant stBAK gene (stBch-2A-stBkt) to create astaxanthin synthesis. The four cassettes to seed-specifically express the B, stB, BAK and stBAK genes were individually combined with a PAC gene (CaPsy-2A-PaCrtI) cassette to previously impart β-carotene-enriched trait in rice endosperm. The single T-DNA vectors of B-PAC, stB-PAC, BAK-PAC and stBAK-PAC resulted in the accumulation of zeaxanthin and astaxanthin in the endosperm of the transgenic rice seeds. In addition, an extended version on the carotenoid pathway was introduced into rice to allow the production of capsanthin, by intercrossing a B-PAC rice line with a Ccs rice line, which harbors a Capsicum capsanthin-capsorubin synthase gene. Ultimately, we developed three functional rice varieties: B-PAC (0.8 μg/g zeaxanthin, deep yellow), stBAK-PAC (1.4 μg/g ketocarotenoids, including astaxanthin, pinkish red) and B-PAC x Ccs (0.4 μg/g of ketoxanthophylls, including capsanthin, orange-red) with the similar levels of total carotenoids to PAC rice, suggesting the capacity was dependent on β-carotene levels. Collectively, a combination of genetic engineering and conventional breeding is effective for multi-step metabolic engineering and biochemical pathway extension.

Topics: beta Carotene; Carotenoids; Crosses, Genetic; Endosperm; Genetic Vectors; Metabolic Engineering; Microarray Analysis; Mixed Function Oxygenases; Oryza; Oxygenases; Plants, Genetically Modified; Polymerase Chain Reaction; Xanthophylls; Zeaxanthins

A major obstacle to industrial-scale astaxanthin production by the yeast Phaffia rhodozyma is the strong inhibitory effect of high glucose concentration on astaxanthin synthesis. We investigated, for the first time, the mechanism of the regulatory effect of high glucose (> 100 g/L) at the metabolite and transcription levels.. Total carotenoid, β-carotene, and astaxanthin contents were greatly reduced in wild-type JCM9042 at high (110 g/L) glucose; in particular, β-carotene content at 24-72 h was only 14-17% of that at low (40 g/L) glucose. The inhibitory effect of high glucose on astaxanthin synthesis appeared to be due mainly to repression of lycopene-to-β-carotene and β-carotene-to-astaxanthin steps in the pathway. Expression of carotenogenic genes crtE, pbs, and ast was also strongly inhibited by high glucose; such inhibition was mediated by creA, a global negative regulator of carotenogenic genes which is strongly induced by glucose. In contrast, astaxanthin-overproducing, glucose metabolic derepression mutant strain MK19 displayed de-inhibition of astaxanthin synthesis at 110 g/L glucose; this de-inhibition was due mainly to deregulation of pbs and ast expression, which in turn resulted from low creA expression. Failure of glucose to induce the genes reg1 and hxk2, which maintain CreA activity, also accounts for the fact that astaxanthin synthesis in MK19 was not repressed at high glucose.. We conclude that astaxanthin synthesis in MK19 at high glucose is enhanced primarily through derepression of carotenogenic genes (particularly pbs), and that this process is mediated by CreA, Reg1, and Hxk2 in the glucose signaling pathway.

Topics: Basidiomycota; beta Carotene; Biosynthetic Pathways; Culture Media; Fungal Proteins; Gene Expression Profiling; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Fungal; Geranylgeranyl-Diphosphate Geranylgeranyltransferase; Glucose; Xanthophylls

In this study, the interaction between four classic dietary antioxidants (including l-ascorbic acid, α-tocopherol, β-carotene and astaxanthin) and papain/bromelain was investigated by fluorescence spectroscopy. The results show that the quenching mechanisms are all static quenching at lower concentrations of antioxidants, but at higher concentrations of antioxidants, predominantly by the "sphere of action" quenching mechanisms. The binding processes of the four antioxidants to papain/bromelain are all synergistically driven by enthalpy and entropy, and the major driving forces are electrostatic effect and hydrophobic interactions. The binding constants of papain/bromelain with the four antioxidants are in the following order as: astaxanthin-papain >β-carotene-papain > astaxanthin-bromelain >l-ascorbic acid-papain >l-ascorbic acid-bromelain >β-carotene-bromelain >α-tocopherol-papain >α-tocopherol-bromelain. Synchronous fluorescence spectroscopy shows the interaction between l-ascorbic acid/β-carotene/astaxanthin and papain/bromelain decreases the hydrophobicity of the microenvironment of tryptophan (Trp) and tyrosine (Tyr) residues. The hydrophobicity of Trp is increased while the hydrophility of Tyr is increased in the presence of α-tocopherol.

Topics: alpha-Tocopherol; Antioxidants; Ascorbic Acid; beta Carotene; Bromelains; Papain; Spectrometry, Fluorescence; Thermodynamics; Tryptophan; Tyrosine; Xanthophylls

Waste deriving from food and agro-industries is a growing problem in our modern society. In order to reduce food waste and its associated impacts over the environment and public health, innovative strategies for the conversion of food wastes in added value products are studied. Whey is one of the prominent waste deriving from Sicilian diary industry. The growth and the carotenoid biosynthesis of the yeast Xanthophyllomyces dendrorhous by its cultivation in pre-treated whey were studied. The study showed the possibility of increasing the astaxanthin productivity from X. dendrorhous in a batch fermentative process under continuous illumination. Our work is focused on the importance of carotenogenesis by X. dendrorhous on whey for the economic and ecological aspect. The results obtained in the study provide useful information about the possible use of this waste material for carotenogenesis from X. dendrorhous.

Topics: Agriculture; Basidiomycota; beta Carotene; Fermentation; Industrial Microbiology; Waste Management; Whey; Xanthophylls

The electronic and vibrational relaxation of carotenoids is one of the key processes in the protection of living cells as well as in the functions of proteins involved in photosynthesis. In this study, the electronic and vibrational relaxation dynamics of β-carotene and its derivatives with substituents on the terminal rings is investigated using femtosecond time-resolved absorption and stimulated Raman spectroscopy in the near-IR region. The carbonyl substituent induces low-frequency shifts of the steady-state and transient absorption bands, decreases of the excited-state lifetimes and the acceleration of vibrational relaxation of the conjugated main chain, whereas the hydroxyl substituent only slightly affects them. The effects of the carbonyl group in the electronic relaxation dynamics are explained well by the lengthening of effective conjugation by the carbonyl group through a partial conjugation between the main chain and the terminal ring. Time-resolved near-IR stimulated Raman spectroscopy demonstrates the significance of the peripheral substitution with the carbonyl group for the vibrational energy relaxation of β-carotene derivatives in the lowest excited singlet state.

Topics: Acetone; beta Carotene; Canthaxanthin; Spectrophotometry; Spectrum Analysis, Raman; Xanthophylls; Zeaxanthins

The unicellular green alga Haematococcus pluvialis accumulates large amounts of the red ketocarotenoid astaxanthin to protect against environmental stresses. Haematococcus cells that accumulate astaxanthin in the central part (green-red cyst cells) respond rapidly to intense light by distributing astaxanthin diffusively to the peripheral part of the cell within 10 min after irradiation. This response is reversible: when astaxanthin-diffused cells were placed in the dark, astaxanthin was redistributed to the center of the cell. Although Haematococcus possesses several pigments other that astaxanthin, the subcellular distribution and content of each pigment remain unknown. Here, we analyzed the subcellular dynamics and localization of major pigments such as astaxanthin, β-carotene, lutein, and chlorophylls under light irradiation using time-lapse and label-free hyperspectral imaging analysis. Fluorescence microscopy and freeze-fracture transmission electron microscopy showed that, preceding/following exposure to light, astaxanthin colocalized with lipid droplets, which moved from the center to the periphery through pathways in a chloroplast. This study revealed that photoresponse dynamics differed between astaxanthin and other pigments (chlorophylls, lutein, and β-carotene), and that only astaxanthin freely migrates from the center to the periphery of the cell through a large, spherical, cytoplasm-encapsulating chloroplast as a lipid droplet. We consider this to be the Haematococcus light-protection mechanism.

Topics: beta Carotene; Carotenoids; Chlorophyceae; Chlorophyll; Chloroplasts; Light; Lipid Droplets; Photosynthesis; Xanthophylls

Optimization of metabolic pathways consisting of large number of genes is challenging. Multivariate modular methods (MMMs) are currently available solutions, in which reduced regulatory complexities are achieved by grouping multiple genes into modules. However, these methods work well for balancing the inter-modules but not intra-modules. In addition, application of MMMs to the 15-step heterologous route of astaxanthin biosynthesis has met with limited success. Here, we expand the solution space of MMMs and develop a multidimensional heuristic process (MHP). MHP can simultaneously balance different modules by varying promoter strength and coordinating intra-module activities by using ribosome binding sites (RBSs) and enzyme variants. Consequently, MHP increases enantiopure 3S,3'S-astaxanthin production to 184 mg l

Topics: beta Carotene; Escherichia coli; Escherichia coli Proteins; Lycopene; Metabolic Engineering; Metabolic Networks and Pathways; Odorants; Xanthophylls

Carotenoids are important phytonutrients with antioxidant properties, and are widely used in foods and feedstuffs as supplements. Astaxanthin, a red-colored ketocarotenoid, has strong antioxidant activity and thus can benefit human health. However, astaxanthin is not produced in most higher plants. Here we report the bioengineering of astaxanthin biosynthesis in rice endosperm by introducing four synthetic genes, sZmPSY1, sPaCrtI, sCrBKT, and sHpBHY, which encode the enzymes phytoene synthase, phytoene desaturase, β-carotene ketolase, and β-carotene hydroxylase, respectively. Transgneic overexpression of two (sZmPSY1 and sPaCrtI), three (sZmPSY1, sPaCrtI and sCrBKT), and all these four genes driven by rice endosperm-specific promoters established the carotenoid/ketocarotenoid/astaxanthin biosynthetic pathways in the endosperm and thus resulted in various types of germplasm, from the yellow-grained β-carotene-enriched Golden Rice to orange-red-grained Canthaxanthin Rice and Astaxanthin Rice, respectively. Grains of Astaxanthin Rice were enriched with astaxanthin in the endosperm and had higher antioxidant activity. These results proved that introduction of a minimal set of four transgenes enables de novo biosynthesis of astaxanthin in the rice endosperm. This work provides a successful example for synthetic biology in plants and biofortification in crops; the biofortified rice products generated by this study could be consumed as health-promoting foods and processed to produce dietary supplements.

Topics: Antioxidants; beta Carotene; Biofortification; Canthaxanthin; Endosperm; Genetic Engineering; Oryza; Plants, Genetically Modified; Xanthophylls

Astaxanthin is a ketocarotenoid produced by photosynthetic microalgae. It is a pigment of high industrial interest in acquaculture, cosmetics, and nutraceutics due to its strong antioxidant power. Haematococcus pluvialis, a fresh-water microalga, accumulates high levels of astaxanthin upon oxidative stress, reaching values up to 5% per dry weight. H. pluvialis accumulates astaxanthin in oil droplets in the cytoplasm, while the chloroplast volume is reduced. In this work, we investigate the biochemical and spectroscopic properties of the H. pluvialis pigment binding complexes responsible for light harvesting and energy conversion. Our findings demonstrate that the main features of chlorophyll and carotenoid binding complexes previously reported for higher plants or Chlamydomonas reinhardtii are preserved under control conditions. Transition to astaxanthin rich cysts however leads to destabilization of the Photosystems. Surprisingly, astaxanthin was found to be bound to both Photosystem I and II, partially substituting β-carotene, and thus demonstrating possible astaxanthin biosynthesis in the plastids or transport from the cytoplasm to the chloroplast. Astaxanthin binding to Photosystems does not however improve their photoprotection, but rather reduces the efficiency of excitation energy transfer to the reaction centers. We thus propose that astaxanthin binding partially destabilizes Photosystem I and II.

Topics: beta Carotene; Carotenoids; Chlorophyll; Chlorophyta; Chloroplasts; Cytoplasm; Light; Photosynthesis; Photosynthetic Reaction Center Complex Proteins; Xanthophylls

Astaxanthin is a high-value ketocarotenoid rarely found in plants. It is derived from β-carotene by the 3-hydroxylation and 4-ketolation of both ionone end groups, in reactions catalyzed by β-carotene hydroxylase and β-carotene ketolase, respectively. We investigated the feasibility of introducing an extended carotenoid biosynthesis pathway into rice endosperm to achieve the production of astaxanthin. This allowed us to identify potential metabolic bottlenecks that have thus far prevented the accumulation of this valuable compound in storage tissues such as cereal grains. Rice endosperm does not usually accumulate carotenoids because phytoene synthase, the enzyme responsible for the first committed step in the pathway, is not present in this tissue. We therefore expressed maize phytoene synthase 1 (ZmPSY1), Pantoea ananatis phytoene desaturase (PaCRTI) and a synthetic Chlamydomonas reinhardtii β-carotene ketolase (sCrBKT) in transgenic rice plants under the control of endosperm-specific promoters. The resulting grains predominantly accumulated the diketocarotenoids canthaxanthin, adonirubin and astaxanthin as well as low levels of monoketocarotenoids. The predominance of canthaxanthin and adonirubin indicated the presence of a hydroxylation bottleneck in the ketocarotenoid pathway. This final rate-limiting step must therefore be overcome to maximize the accumulation of astaxanthin, the end product of the pathway.

Topics: beta Carotene; Chlamydomonas reinhardtii; Endosperm; Genetic Engineering; Geranylgeranyl-Diphosphate Geranylgeranyltransferase; Metabolic Engineering; Metabolic Networks and Pathways; Mixed Function Oxygenases; Oryza; Oxidoreductases; Oxygenases; Plants, Genetically Modified; Xanthophylls; Zea mays

L-Ascorbic acid, α-tocopherol, procyanidin B3, β-carotene and astaxanthin are five classic dietary antioxidants. In this study, the interaction between the five antioxidants and ovalbumin was investigated by fluorescence spectroscopy, in combination with UV-vis absorption spectroscopy and circular dichroism (CD) spectroscopy. The quenching mechanism of ovalbumin by α-tocopherol is static quenching and the interaction between α-tocopherol and ovalbumin is synergistically driven by enthalpy and entropy. Electrostatic interactions and hydrophobic interactions play a major role in stabilizing the complex. For the other four antioxidants, the quenching mechanisms are all static quenching mechanisms at lower concentrations of antioxidants, but at higher concentrations of antioxidants, predominantly by the "sphere of action" quenching mechanisms. The binding processes of the other four antioxidants to ovalbumin are all entropy process and the major part of the action force is hydrophobic interactions. The binding constants of ovalbumin with the five antioxidants are in the following order as: astaxanthin > β-carotene > L-ascorbic acid > procyanidin B3 > α-tocopherol at 298 K. Synchronous fluorescence spectroscopy shows the interaction between L-ascorbic acid/β-carotene/astaxanthin and ovalbumin decreases the hydrophobicity of the microenvironment of tryptophan (Trp) and tyrosine (Tyr) residues. The hydrophobicity of Trp is increased while the hydrophility of Tyr is increased in the presence of α-tocopherol. However, the microenvironment of Trp and Tyr is not affected by procyanidin B3. The UV-vis absorption and CD spectra suggest that the interaction between the five antioxidants and ovalbumin leads to the loosening and unfolding of ovalbumin skeleton and exerts some influence on the natural secondary structure of ovalbumin. The study provides an accurate and full basic data for clarifying the binding mechanisms of L-ascorbic acid, α-tocopherol, procyanidin B3, β-carotene and astaxanthin interacting with ovalbumin and is helpful for understanding rational use of antioxidants as dietary supplements.

Topics: alpha-Tocopherol; Animals; Antioxidants; Ascorbic Acid; beta Carotene; Biflavonoids; Binding Sites; Catechin; Circular Dichroism; Humans; Ovalbumin; Proanthocyanidins; Protein Binding; Spectrometry, Fluorescence; Thermodynamics; Xanthophylls

Ketolated and hydroxylated carotenoids are high-value compounds with industrial, food, and feed applications. Chemical synthesis is currently the production method of choice for these compounds, with no amenable plant sources readily available. In this study, the 4,4' β-oxygenase (

Topics: Bacterial Proteins; beta Carotene; Biosynthetic Pathways; Carotenoids; Caulobacteraceae; Flowers; Gene Expression; Microscopy, Electron, Transmission; Mixed Function Oxygenases; Molecular Structure; Nicotiana; Oxygenases; Plant Leaves; Plants, Genetically Modified; Plastids; Reverse Transcriptase Polymerase Chain Reaction; Thylakoids; Xanthophylls

Highly efficient biosynthesis of the commercially valuable carotenoid astaxanthin by microbial cells is an attractive alternative to chemical synthesis and microalgae extraction. With the goal of enhancing heterologous astaxanthin production in Saccharomyces cerevisiae, metabolic engineering and protein engineering were integrated to improve both the expression and activity of rate-limiting enzymes. Firstly, to increase the supply of β-carotene as a key precursor for astaxanthin, a positive mutant of GGPP synthase (CrtE03M) was overexpressed together with three other rate-limiting enzymes tHMG1, CrtI and CrtYB. Subsequently, to accelerate the conversion of β-carotene to astaxanthin, a color screening system was developed and adopted for directed evolution of β-carotene ketolase (OBKT), generating a triple mutant OBKTM (H165R/V264D/F298Y) with 2.4-fold improved activity. After adjusting copy numbers of the above-mentioned rate-limiting enzymes to further balance the metabolic flux, a diploid strain YastD-01 was generated by mating two astaxanthin-producing haploid strains carrying the same carotenogenic pathway. Finally, further overexpression of OCrtZ and OBKTM in YastD-01 resulted in accumulation of 8.10mg/g DCW (47.18mg/l) of (3S, 3'S)-astaxanthin in shake-flask cultures. This combinatorial strategy might be also applicable for alleviation of metabolic bottleneck in biosynthesis of other value-added products, especially colored metabolites.

Topics: beta Carotene; Biomass; Biosynthetic Pathways; Diploidy; Directed Molecular Evolution; Farnesyltranstransferase; Fermentation; Industrial Microbiology; Metabolic Engineering; Mutant Proteins; Oxygenases; Protein Engineering; Recombinant Proteins; Saccharomyces cerevisiae; Volvocida; Xanthophylls

β-Carotene and astaxanthin are two carotenoids with powerful antioxidant properties, but the binding mechanisms of β-carotene/astaxanthin to proteases remain unclear. In this study, the interaction of these two carotenoids with trypsin and pepsin was investigated using steady-state and time-resolved fluorescence measurements, synchronous fluorescence spectroscopy, UV-vis absorption spectroscopy and circular dichroism (CD) spectroscopy. The experimental results indicated that the quenching mechanisms of trypsin/pepsin by the two carotenoids are static processes. The binding constants of trypsin and pepsin with these two carotenoids are in the following order: astaxanthin-trypsin > astaxanthin-pepsin > β-carotene-trypsin > β-carotene-pepsin, respectively. Thermodynamic investigations revealed that the interaction between the two carotenoids and trypsin/pepsin is synergistically driven by enthalpy and entropy, and hydrophobic forces and electrostatic attraction have a significant role in the reactions. In addition, as shown by synchronous fluorescence spectroscopy, UV-vis absorption spectroscopy and CD, the two carotenoids may induce conformational and microenvironmental changes in trypsin/pepsin. The study provides an accurate and full basic data for clarifying the binding mechanisms of the two carotenoids with trypsin/pepsin and is helpful in understanding their effect on protein function and their biological activity in vivo.

Topics: beta Carotene; Circular Dichroism; Pepsin A; Spectrometry, Fluorescence; Spectrophotometry, Ultraviolet; Thermodynamics; Trypsin; Xanthophylls

Astaxanthin, a red C40 carotenoid, is one of the most abundant marine carotenoids. It is currently used as a food and feed additive in a hundred-ton scale and is furthermore an attractive component for pharmaceutical and cosmetic applications with antioxidant activities. Corynebacterium glutamicum, which naturally synthesizes the yellow C50 carotenoid decaprenoxanthin, is an industrially relevant microorganism used in the million-ton amino acid production. In this work, engineering of a genome-reduced C. glutamicum with optimized precursor supply for astaxanthin production is described. This involved expression of heterologous genes encoding for lycopene cyclase CrtY, β-carotene ketolase CrtW, and hydroxylase CrtZ. For balanced expression of crtW and crtZ their translation initiation rates were varied in a systematic approach using different ribosome binding sites, spacing, and translational start codons. Furthermore, β-carotene ketolases and hydroxylases from different marine bacteria were tested with regard to efficient astaxanthin production in C. glutamicum. In shaking flasks, the C. glutamicum strains developed here overproduced astaxanthin with volumetric productivities up to 0.4 mg·L(-1)·h(-1) which are competitive with current algae-based production. Since C. glutamicum can grow to high cell densities of up to 100 g cell dry weight (CDW)·L(-1), the recombinant strains developed here are a starting point for astaxanthin production by C. glutamicum.

Topics: beta Carotene; Carotenoids; Corynebacterium glutamicum; Metabolic Engineering; Mixed Function Oxygenases; Oxygenases; Xanthophylls

The experiment was conducted to determine the most effective antioxidant (among the vitamin E (VE), vitamin C (VC), vitamin A (VA), astaxanthine (AX), β-carotene (βC)) on the ovarian egg number and size, level of VE, VC, VA, AX, βC and oxidative stress (as malondialdehyde (MDA)) in the hepatopancreas, ovarian, gills and muscle tissue during ovarian development of Astacus leptodactylus. One control (C) and five experimental diets (EE, EC, EA, EAX and EβC) were prepared. The EE, EC, EA, EAX and EβC groups were formed by added 150 mg kg-1 VE, 200 mg kg-1 VC, 240 mg kg-1 VA, 200 mg kg-1 AX and 200 mg kg-1 βC to diet C, respectively. At the end of the experiment found that the dietary antioxidants increased ovarian egg number and size and reduced the level of MDA in the tissues. Ovarian egg number and size were highest in the EE and EAX diet groups in the comparison to control (p<0.001). The level of MDA in the tissues was lowest in the EAX diet group in the comparison to control (p<0.001). The highest levels of VE, VC, VA, AX and βC were found in the hepatopancreas and ovarian compared with muscle and gills. The highest level of MDA also was determined in the ovarian according to other tissues. In conclusion, the VE and AX in broodstock diets were the most effective antioxidants on the ovarian egg number and size of A. leptodactylus.

Topics: Animals; Antioxidants; Ascorbic Acid; Astacoidea; beta Carotene; Cell Count; Cell Size; Chromatography, High Pressure Liquid; Dietary Supplements; Female; Malondialdehyde; Oocytes; Ovary; Oxidative Stress; Reproduction; Vitamin A; Vitamin E; Vitamins; Xanthophylls

β-Carotene and astaxanthin are two carotenoids with powerful antioxidant properties. In this study, the interaction of these two carotenoids with human serum albumin (HSA) and bovine serum albumin (BSA) under physiological conditions was investigated using several spectroscopic techniques. The experimental results indicate the quenching mechanism of HSA/BSA, by the two carotenoids, is a static process. The binding constants and number of binding sites were evaluated at different temperatures. Thermodynamic investigations revealed the interaction between the two carotenoids and HSA/BSA is synergistically driven by enthalpy and entropy, and hydrophobic forces and electrostatic attraction have a significant role in the reactions. Binding site I was found to be the primary binding site for β-carotene and astaxanthin. In addition, as shown by synchronous fluorescence spectroscopy and FT-IR, the two carotenoids may induce conformational and micro-environmental changes in HSA/BSA.

Topics: beta Carotene; Binding Sites; Humans; Protein Binding; Serum Albumin; Serum Albumin, Bovine; Spectrometry, Fluorescence; Spectroscopy, Fourier Transform Infrared; Thermodynamics; Xanthophylls

This study was conducted to investigate the effects of two carotenoids (astaxanthin and β-carotene) on the sperm quality of goldfish Carassius auratus (Linnaeus, 1758). For this purpose, six diets containing concentrations of 50, 100, and 150 mg/kg of synthetic astaxanthin and β-carotene were added to a basic carp diet. One group of fish was also fed with a control diet (no added carotenoids). Osmolality, spermatocrit value, and sperm concentration significantly increased in the treatment supplemented with 150 mg/kg of astaxanthin (296.6 ± 1.1 mOsm/kg; 29.2 ± 0.6%; 17.2 ± 0.4 × 10(9) cells/mL, respectively) and β-carotene (295.2 ± 2.1 mOsm/kg; 32.5 ± 1.6%; 17.9 ± 0.5 × 10(9) cells/mL, respectively). The highest concentration of astaxanthin (10.4 ± 1.4 mg/kg) was recorded in the treatment of A150 (P < 0.05) and did not differ between β-carotene treatments. The highest motility was observed in the A150 and B150 treatments, and the lowest was observed in the control group (P < 0.05). The artificial fertilization of the treated males with the similar females (fed with the control diet) showed that the fertilization rate in the A150 treatments was higher than in the other treatments (P < 0.05). In conclusion, dietary supplementation with 150 mg/kg of astaxanthin improves osmolality, motility, fertilization rate, and sperm concentration.

Topics: Animals; beta Carotene; Diet; Dietary Supplements; Dose-Response Relationship, Drug; Female; Fertilization; Goldfish; Male; Osmolar Concentration; Sperm Count; Sperm Motility; Spermatozoa; Xanthophylls

Carotenoids prevent different degenerative diseases and improve human health. Microalgae are commercially exploited for carotenoids, including astaxanthin and β-carotene. Two commercially important microalgae, Dunaliella salina and Tetraselmis suecica, were treated with plant hormones salicylic acid (SA) and methyl jasmonate (MJ), or by UV-C radiation (T. suecica only) and a combination thereof. Significant increases in total carotenoids were found for D. salina and T. suecica after treatment with MJ (10 μmol/L) and SA (70-250 μmol/L), respectively. T. suecica also had significant increases in total carotenoids following UV-C radiation compared to control cultures. Among the carotenoids, lutein was the highest induced carotenoid. A combination of these two treatments also showed a significant increase in total carotenoids and lutein for T. suecica, when compared to controls. Plant hormones and UV-C radiation may be useful tools for increasing carotenoid accumulation in green microalgae although the responses are species- and dose-specific and should be trialed in medium to large scale to explore commercial production.

Topics: Acetates; Anti-Infective Agents; beta Carotene; Carotenoids; Chlorophyta; Cyclopentanes; Lutein; Microalgae; Oxylipins; Plant Growth Regulators; Salicylic Acid; Ultraviolet Rays; Water Microbiology; Xanthophylls

A simple and accurate reversed phase high-performance liquid chromatography coupled with diode array detector (HPLC-DAD) method for simultaneously determining and quantifying the antioxidants carotenes, xanthophylls, and retinol in human plasma is presented in this paper. Compounds were extracted with hexane, a C30 column, and a mobile phase of methanol, methyl tert-butyl ether, and water were used for the separation of the compounds. A total of 8 carotenoids, 3 Z-β-carotene isomers, and 1 fat-soluble vitamin (retinol) were resolved within 72 min at a flow rate of 0.6 mL/min. Detection was achieved at 450 nm for carotenoids and 330 nm for retinol. To evaluate the effectiveness of themethod, it has been applied to an intervention study conducted on eight volunteers. Results. Limits of detection were between 0.1 μg/mL for lycopene and astaxanthin and 1.3 μg/mL for 15-Z-β-carotene. Recoveries were ranged between 89% and 113% for α-carotene and astaxanthin, respectively. Accuracy was between 90.7% and 112.2% and precision was between 1% and 15% RSD. In human plasma samples compounds studied were identified besides three lycopene isomers, demonstrated to be suitable for application in dietary intervention studies. Conclusions. Due to its accuracy, precision, selectivity, and reproducibility, this method is suitable to dietary habits and/or antioxidants status studies.

Topics: Antioxidants; beta Carotene; Carotenoids; Chromatography, High Pressure Liquid; Humans; Limit of Detection; Lycopene; Vitamin A; Xanthophylls

In recent years, astaxanthin is claimed to have a 10 times higher antioxidant activity than that of other carotenoids such as lutein, zeaxanthin, canthaxanthin, and β-carotene; the antioxidant activity of astaxanthin is 100 times higher than that of α-tocopherol. Penaeus monodon (tiger shrimp) is the largest commercially available shrimp species and its waste is a rich source of carotenoids such as astaxanthin and its esters. The efficient and environment-friendly recovery of astaxanthins was accomplished by using a supercritical fluid extraction (SFE) technique. The effects of different co-solvents and their concentrations on the yield and composition of the extract were investigated. The following co-solvents were studied prior to the optimization of the SFE technique: ethanol, water, methanol, 50% (v/v) ethanol in water, 50% (v/v) methanol in water, 70% (v/v) ethanol in water, and 70% (v/v) methanol in water. The ethanol extract produced the highest carotenoid yield (84.02 ± 0.8 μg/g) dry weight (DW) with 97.1% recovery. The ethanol extract also produced the highest amount of the extracted astaxanthin complex (58.03 ± 0.1 μg/g DW) and the free astaxanthin content (12.25 ± 0.9 μg/g DW) in the extract. Lutein and β-carotene were the other carotenoids identified. Therefore, ethanol was chosen for further optimization studies.

Topics: Animals; beta Carotene; Canthaxanthin; Chromatography, Supercritical Fluid; Ethanol; Lutein; Penaeidae; Solid Waste; Solvents; Xanthophylls

Newly identified spore-forming pigmented marine bacteria, Bacillus indicus HU36 and Bacillus firmus GB1, are sources of carotenoids (mainly 15 yellow and orange pigments and 13 pink pigments, respectively) with original structures. These bacterial carotenoids were evaluated for their ability to inhibit the iron-induced peroxidation of linoleic acid micelles, or sunflower oil-in-water emulsions, in comparison with β-carotene, lycopene and astaxanthin. Lipid peroxidation was carried out in acidic conditions and initiated by dietary heme or non-heme iron (metmyoglobin or Fe(II), respectively) so as to simply simulate the postprandial gastric medium, a possible site for dietary oxidative stress. Lipid hydroperoxide formation and carotenoid consumption were followed by UV-vis spectroscopy and appropriate indicators of the antioxidant activity were estimated in each model. The bacterial carotenoids were found to be better inhibitors of heme-induced lipid peroxidation than the reference carotenoids as a likely consequence of their location closer to the interface in micelles and lipid droplets. However, this trend was not confirmed in lipid peroxidation induced by non-heme iron, possibly because of the redox recycling of Fe(II) by carotenoids. The quantitative kinetic analysis of the peroxidation curves suggests that the carotenoids mainly inhibit the propagation phase of lipid peroxidation by direct scavenging of the lipid peroxyl radicals, in agreement with independent experiments showing that carotenoids are unable to reduce the one-electron oxidized form of metmyoglobin (ferrylmyoglobin), a model of initiating species in heme-induced lipid peroxidation. Overall, carotenoids from Bacillus indicus HU36 and Bacillus firmus GB1 were found to be interesting antioxidants to fight postprandial oxidative stress in the stomach.

Topics: Antioxidants; Bacillus; beta Carotene; Carotenoids; Gastric Mucosa; Humans; Iron; Linoleic Acid; Lipid Peroxidation; Lycopene; Metmyoglobin; Micelles; Models, Theoretical; Oxidation-Reduction; Oxidative Stress; Peroxides; Postprandial Period; Stomach; Xanthophylls

The objective of this study was to determine chemiluminescence (CL) antioxidant activities and neuroprotective effects of astaxanthin, beta-carotene (β-carotene), and canthaxanthin on undifferentiated rat pheochromocytoma (PC12) cells. We performed three CL antioxidant assays, and the three carotenoids showed varying degrees of antioxidant activity, with astaxanthin exhibiting the highest antioxidant activity than the other two samples. Results of a pyrogallol-luminol assay revealed β-carotene to have higher antioxidant activity than canthaxanthin, whereas cupric sulfate-Phen-Vc-hydrogen peroxide (H₂O₂) assay showed canthaxanthin to have higher antioxidant activity than β-carotene. Luminol-H₂O₂ assay showed the antioxidant activity series as canthaxanthin > β-carotene at 62.5-1000 μg/mL and β-carotene > canthaxanthin at 1000-4000 μg/mL. Astaxanthin exhibited partial neuroprotective activity against H₂O₂ and the strongest neuroprotective activity against amyloid beta-peptide(25-35) [(Aβ)(25-35)]-induced undifferentiated PC12 cell deaths at 0.5-5.0 μM. Canthaxanthin showed partial neuroprotective activity in Aβ(25-35)-induced undifferentiated PC12 cell deaths at 1.0-5.0 μM. Astaxanthin protected undifferentiated PC12 cells from the damaging effects of H₂O₂ and Aβ(25-35) by the following ways: (1) scavenging superoxide anion radicals, hydroxyl radicals, and H₂O₂; (2) securing cell viability; (3) suppressing the production of reactive oxygen species; and (4) eliminating calcium ion influx. Our results conclusively show that astaxanthin has the merit as a potential neuron protectant.

Topics: Animals; Antioxidants; beta Carotene; Canthaxanthin; Luminescence; Neurons; Neuroprotective Agents; PC12 Cells; Rats; Reactive Oxygen Species; Vitamins; Xanthophylls

In this study, low temperature vacuum microwave-assisted extraction, which simultaneous performed microwave-assisted extraction (MAE) in low temperature and in vacuo environment, was proposed. The influencing parameters including solid/liquid ratio, extraction temperature, extraction time, degree of vacuum and microwave power were discussed. The predominance of low temperature vacuum microwave-assisted extraction was investigated by comparing the extraction yields of vitamin C, β-carotene, aloin A and astaxanthin in different foods with that in MAE and solvent extraction, and 5.2-243% increments were obtained. On the other hand, the chemical kinetics of vitamin C and aloin A, which composed two different steps including the extraction step of analyte transferred from matrix into solvent and the decomposition step of analyte degraded in the extraction solvent, were proposed. All of the decomposition rates (K(2)) for the selected analyte in low temperature, in vacuo and in nitrogen atmosphere decreased significantly comparing with that in conventional MAE, which are in agreement with that obtained from experiments. Consequently, the present method was successfully applied to extract labile compound from different food samples. These results showed that low temperature and/or in vacuo environment in microwave-assisted extraction system was especially important to prevent the degradation of labile components and have good potential on the extraction of labile compound in foods, pharmaceutical and natural products.

Topics: Ascorbic Acid; beta Carotene; Chromatography, High Pressure Liquid; Cold Temperature; Emodin; Food Analysis; Kinetics; Microwaves; Xanthophylls

Carotenoids and their metabolites are essential factors for the maintenance of important life processes such as photosynthesis. Animals cannot synthesize carotenoids de novo, they must obtain them via their food. In order to make intensive animal husbandry possible and maintain human and animal health synthetic nature identical carotenoids are presently commercially available at the multi-tonnes scale per year. Synthetically accessible (13)C enriched carotenoids are essential to apply isotope sensitive techniques to obtain information at the atomic level without perturbation about the role of carotenoids in photosynthesis, nutrition, vision, animal development, etc. Simple highly (13)C enriched C(1), C(2) and C(3) building blocks are commercially available via 99% (13)CO. The synthetic routes for the preparation of the (13)C enriched building blocks starting from the commercially available systems are discussed first. Then, how these building blocks are used for the synthesis of the various (13)C enriched carotenoids and apocarotenoids are reviewed next. The synthetic Schemes that resulted in (13)C enriched β-carotene, spheroidene, β-cryptoxanthin, canthaxanthin, astaxanthin, (3R,3'R)-zeaxanthin and (3R,3'R,6'R)-lutein are described. The Schemes that are reviewed can also be used to synthetically access any carotenoid and apocarotenoid in any (13)C isotopically enriched form up to the unitarily enriched form.

Topics: beta Carotene; Canthaxanthin; Carbon Isotopes; Carotenoids; Molecular Structure; Stereoisomerism; Xanthophylls; Zeaxanthins

Since the 1980's when the predominate focus of study and use of carotenoids in human nutritional formulations was solely on beta-carotene, there has been a steady increase in research aimed to understand the role of a wide variety of carotenoids in human health. This work has increasingly demonstrated the benefits of a number of carotenoids, and there has been a corresponding increase in the number of carotenoids provided in nutritional supplements (multicarotenoids). Numerous published observations in both human and animal studies suggest significant interaction and competition between various carotenoids during absorption and metabolism, resulting in the inhibition of uptake of one over the other. This competition has the end result of reducing the beneficial effects of the inhibited carotenoid. To limit such competition and maximize carotenoid uptakes, a layered beadlet was designed to release a defined ratio of carotenoids sequentially. Preliminary dissolution testing is presented showing the release profile in simulated digestive conditions of a combination of beta-carotene, alpha carotene, lutein, zeaxanthin, lycopene and astaxanthin derived from natural sources. Comparison is made to an immediate release beadlet formulation using the same combination of carotenoids. These results will be used to guide proof of concept clinical testing for effectiveness in humans.

Topics: beta Carotene; Carotenoids; Chemistry, Pharmaceutical; Chromatography, High Pressure Liquid; Dietary Supplements; Humans; Lutein; Lycopene; Solubility; Xanthophylls; Zeaxanthins

Several fungal species, particularly some included in the Mucorales, have been used to develop fermentation processes for the production of β-carotene. Oxygenated derivatives of β-carotene are more valuable products, and the preference by the market of carotenoids from biological sources has increased the research in different carotenoid-producing organisms. We currently use Mucor circinelloides as a model organism to develop strains able to produce new, more valuable, and with an increased content of carotenoids. In this chapter we describe part of our efforts to construct active gene fusions which could advance in the diversification of carotenoid production by this fungus. The main carotenoid accumulated by M. circinelloides is β-carotene, although it has some hydroxylase activity and produces low amounts of zeaxanthin. Two enzymatic activities are required for the production of astaxanthin from β-carotene: a hydroxylase and a ketolase. We used the ctrW gene of Paracoccus sp. N81106, encoding a bacterial β-carotene ketolase, to construct gene fusions with two fungal genes essential for the modification of the pathway in M. circinelloides. First we fused it to the carRP gene of M. circinelloides, which is responsible for the phytoene synthase and lycopene cyclase activities in this fungus. The expected activity of this fusion gene would be the accumulation by M. circinelloides of canthaxanthin and probably some astaxanthin. A second construction was the fusion of the crtW gene of Paracoccus sp. to the crtS gene of Xanthophyllomyces dendrorhous, responsible for the synthesis of astaxanthin from β-carotene in this fungus, but which was shown to have only hydroxylase activity in M. circinelloides. The expected result in M. circinelloides transformants was the accumulation of astaxanthin. Here we describe a detailed and empirically tested protocol for the construction of these gene fusions.

Topics: Artificial Gene Fusion; beta Carotene; Carotenoids; DNA, Complementary; DNA, Fungal; Genes, Fungal; Mucor; RNA, Fungal; Transformation, Genetic; Xanthophylls

Carotenoid bioavailability is affected by numerous factors. Our aim was to assess the involvement of known carotenoid physicochemical properties (e.g., hydrophobicity, van der Waals volume,…) on the transport of the main dietary carotenoids (β-carotene, lycopene, lutein, and astaxanthin, from their food matrix to their main storage tissues.. We used four complementary models: synthetic mixed micelles, an in vitro digestion procedure, Caco-2 cell monolayers, and a gavage experiment in rats. The efficiency with which pure carotenoids were incorporated into synthetic mixed micelles was related to their melting points (r = 0.99, p = 0.015). The efficiency with which pure carotenoids were transferred from dietary triglycerides into mixed micelles was related to carotenoid hydrophobicity (r = -1, p = 0.005). There was no relationship between the carotenoid physicochemical properties studied and their uptake efficiency by Caco-2. The postprandial plasma carotenoid response to carotenoid gavage was related to carotenoid hydrophobicity (r = -0.99, p = 0.006). Carotenoid adipose tissue response was not related to the carotenoid physicochemical properties studied.. Thus, carotenoid hydrophobicity is important for bioaccessibility and postprandial blood response of carotenoids. In contrast, the carotenoid physicochemical properties studied are apparently not strong determinants of carotenoid uptake by enterocytes and adipose tissue.

Topics: Adipose Tissue; Animals; beta Carotene; Biological Availability; Caco-2 Cells; Carotenoids; Diet; Humans; Intestinal Absorption; Intestinal Mucosa; Intestines; Liver; Lutein; Lycopene; Male; Micelles; Models, Animal; Postprandial Period; Rats; Rats, Wistar; Triglycerides; Xanthophylls

Xanthophyllomyces dendrorhous is a natural source of astaxanthin, a carotenoid widely used in the food industry. In this yeast, astaxanthin is synthesized from β-carotene by a cytochrome P450, CrtS, which depends on CrtR, the four-domain cytochrome P450 reductase (CPR). Although Saccharomyces cerevisiae has an endogenous CPR (ScCPR), expression of CrtS does not result in astaxanthin production unless it is coexpressed with CrtR. Assuming that CrtS could interact with the FMN-binding domain of either CrtR or ScCPR (XdFMNbd and ScFMNbd, respectively), the aim of this work was to identify possible interaction differences between these alternative complexes by protein modeling and short molecular dynamics simulations. Considering the recently proposed membrane orientation of a mammalian P450, our CrtS-CrtR model predicts that both N-terminal ends stand adjacent to the membrane plane, allowing their anchoring. Compared with the possible interface between CrtS and both FMNbd, the Xanthophyllomyces system appears to be stabilized by more saline bridges.

Topics: Basidiomycota; beta Carotene; Binding Sites; Cytochrome P-450 Enzyme System; Flavin Mononucleotide; Models, Molecular; NADPH-Ferrihemoprotein Reductase; Protein Structure, Secondary; Xanthophylls

The isolation and characterization of the lycopene ε-cyclase gene from the green microalga Chlorella (Chromochloris) zofingiensis (Czlcy-e) was performed. This gene is involved in the formation of the carotenoids α-carotene and lutein. Czlcy-e gene encoded a polypeptide of 654 amino acids. A single copy of Czlcy-e was found in C. zofingiensis. Functional analysis by heterologous complementation in Escherichia coli showed the ability of this protein to convert lycopene to δ-carotene. In addition, the regulation of the carotenogenic pathway by light and nitrogen was also studied in C. zofingiensis. High irradiance stress did not increase mRNA levels of neither lycopene β-cyclase gene (lcy-b) nor lycopene ε-cyclase gene (lcy-e) as compared with low irradiance conditions, whereas the transcript levels of psy, pds, chyB and bkt genes were enhanced, nevertheless triggering the synthesis of the secondary carotenoids astaxanthin, canthaxanthin and zeaxanthin and decreasing the levels of the primary carotenoids α-carotene, lutein, violaxanthin and β-carotene. Nitrogen starvation per se enhanced mRNA levels of all genes considered, except lcy-e and pds, but did not trigger the synthesis of astaxanthin, canthaxanthin nor zeaxanthin. The combined effect of both high light and nitrogen starvation stresses enhanced significantly the accumulation of these carotenoids as well as the transcript levels of bkt gene, as compared with the effect of only high irradiance stress.

Topics: beta Carotene; Canthaxanthin; Carotenoids; Chlorella; Escherichia coli; Intramolecular Lyases; Light; Lutein; Microalgae; Nitrogen; RNA, Messenger; Stress, Physiological; Transcription, Genetic; Xanthophylls; Zeaxanthins

Hydroxyl radical reacts readily with β-carotene following submicrosecond laser photolysis of N-hydroxypyridine-2(1H)-thione (N-HPT) as a "photo-Fenton" reagent generating hydroxyl and thiyl radicals in acetonitrile:tetrahydrofuran (4:1, v/v) solution. On the basis of spectral evidence, and supported by kinetic considerations and thermodynamic calculations, a short-lived transient species, detected by time-resolved absorption spectroscopy with an absorption maximum at ∼750 nm and a lifetime of ∼150 ns at 25 °C under anaerobic conditions, is suggested to be the long-sought neutral β-carotene radical formed by hydrogen-atom abstraction. The transient spectrum is different from the spectra of the β-carotene radical cation (∼1000 nm absorption maximum with a millisecond lifetime), the β-carotene radical adducts (∼520 nm, several microsecond lifetime), the β-carotene radical cation ion pair (∼750 nm, several hundred microsecond lifetime), and the β-carotene radical anion (∼880 nm, a few tens of microsecond lifetime). In parallel, β-carotene reacts with the thiyl radical to yield a sulfur radical adduct with absorption maximum at ∼520 nm with a lifetime of 3.0 μs. For astaxanthin and canthaxanthin, the reaction with the thiyl radical dominates and the neutral radical is hardly formed in agreement with the less reducing properties of these 4,4'-diketo carotenoids without the reactive 4,4'-hydrogens.

Topics: Absorption; beta Carotene; Canthaxanthin; Hydroxyl Radical; Lasers; Photolysis; Pyridines; Spectrum Analysis; Thiones; Xanthophylls

Carotenoids have potent antioxidant activity as well as therapeutic value, and their formation has been seen to be induced in algae by stress, including high-salt culture conditions. A differential profiling of carotenoids was conducted using a targeted metabolomics approach with accurate mass data generated by liquid chromatography-electrospray-time-of-flight (LC-ESI-TOF) mass spectrometry followed by postacquisition filtering based on isotope patterns and mass defects to detect carotenoids up-regulated in Scenedesmus sp. exposed to high-salt conditions. Algal cultures treated with high concentrations of sodium acetate or sodium chloride were found to cause an increase in various carotenoids. On the basis of differential analysis, astaxanthin and canthaxanthin increased upon salt treatment. Astaxanthin, in its free form and as fatty acid esters, was seen to increase in Scenedesmus sp. using accurate mass MS. A few other carotenoid compounds increased upon salt treatment, including echinenone and adonirubin, involved in the pathway of astaxanthin biosynthesis from β-carotene, as well as isomers of astaxanthin and canthaxanthin. A time course study of acetate treatment was done to observe the time-dependent up-regulation of carotenogenesis.

Topics: beta Carotene; Canthaxanthin; Carotenoids; Chromatography, Liquid; Lutein; Mass Spectrometry; Metabolomics; Scenedesmus; Sodium Acetate; Sodium Chloride; Up-Regulation; Xanthophylls

The xanthophyll astaxanthin is a high-value compound with applications in the nutraceutical, cosmetic, food, and animal feed industries. Besides chemical synthesis and extraction from naturally producing organisms like Haematococcus pluvialis, heterologous biosynthesis in non-carotenogenic microorganisms like Escherichia coli, is a promising alternative for sustainable production of natural astaxanthin. Recent achievements in the metabolic engineering of E. coli strains have led to a significant increase in the productivity of carotenoids like lycopene or β-carotene by increasing the metabolic flux towards the isoprenoid precursors. For the heterologous biosynthesis of astaxanthin in E. coli, however, the conversion of β-carotene to astaxanthin is obviously the most critical step towards an efficient biosynthesis of astaxanthin.. Here we report the construction of the first plasmid-free E. coli strain that produces astaxanthin as the sole carotenoid compound with a yield of 1.4 mg/g cdw (E. coli BW-ASTA). This engineered E. coli strain harbors xanthophyll biosynthetic genes from Pantoea ananatis and Nostoc punctiforme as individual expression cassettes on the chromosome and is based on a β-carotene-producing strain (E. coli BW-CARO) recently developed in our lab. E. coli BW-CARO has an enhanced biosynthesis of the isoprenoid precursor isopentenyl diphosphate (IPP) and produces β-carotene in a concentration of 6.2 mg/g cdw. The expression of crtEBIY along with the β-carotene-ketolase gene crtW148 (NpF4798) and the β-carotene-hydroxylase gene (crtZ) under controlled expression conditions in E. coli BW-ASTA directed the pathway exclusively towards the desired product astaxanthin (1.4 mg/g cdw).. By using the λ-Red recombineering technique, genes encoding for the astaxanthin biosynthesis pathway were stably integrated into the chromosome of E. coli. The expression levels of chromosomal integrated recombinant biosynthetic genes were varied and adjusted to improve the ratios of carotenoids produced by this E. coli strain. The strategy presented, which combines chromosomal integration of biosynthetic genes with the possibility of adjusting expression by using different promoters, might be useful as a general approach for the construction of stable heterologous production strains synthesizing natural products. This is the case especially for heterologous pathways where excessive protein overexpression is a hindrance.

Topics: beta Carotene; Chromosomes; Escherichia coli; Genetic Engineering; Mixed Function Oxygenases; Nostoc; Oxygenases; Pantoea; Plasmids; Xanthophylls

A few species in the genus Adonis are the only land plants known to produce the valuable red ketocarotenoid astaxanthin in abundance. Here, we ascertain the pathway that leads from the β-rings of β-carotene, a carotenoid ubiquitous in plants, to the 3-hydroxy-4-keto-β-rings of astaxanthin (3,3'-dihydroxy-β,β-carotene-4,4'-dione) in the blood-red flowers of Adonis aestivalis, an ornamental and medicinal plant commonly known as summer pheasant's eye. Two gene products were found to catalyze three distinct reactions, with the first and third reactions of the pathway catalyzed by the same enzyme. The pathway commences with the activation of the number 4 carbon of a β-ring in a reaction catalyzed by a carotenoid β-ring 4-dehydrogenase (CBFD), continues with the further dehydrogenation of this carbon to yield a carbonyl in a reaction catalyzed by a carotenoid 4-hydroxy-β-ring 4-dehydrogenase, and concludes with the addition of an hydroxyl group at the number 3 carbon in a reaction catalyzed by the erstwhile CBFD enzyme. The A. aestivalis pathway is both portable and robust, functioning efficiently in a simple bacterial host. Our elucidation of the pathway to astaxanthin in A. aestivalis provides enabling technology for development of a biological production process and reveals the evolutionary origin of this unusual plant pathway, one unrelated to and distinctly different from those used by bacteria, green algae, and fungi to synthesize astaxanthin.

Topics: Adonis; Base Sequence; beta Carotene; Escherichia coli; Evolution, Molecular; Flowers; Gene Library; Genetic Complementation Test; Molecular Sequence Data; Oxidoreductases; Phylogeny; Plant Proteins; Plants, Medicinal; Sequence Analysis, DNA; Xanthophylls

Haematococcus pluvialis is a freshwater unicellular green microalga belonging to the class Chlorophyceae and is of commercial interest for its ability to accumulate massive amounts of the red ketocarotenoid astaxanthin (3,3'-dihydroxy-β,β-carotene-4,4'-dione). Using confocal Raman microscopy and multivariate analysis, we demonstrate the ability to spectrally resolve resonance-enhanced Raman signatures associated with astaxanthin and β-carotene along with chlorophyll fluorescence. By mathematically isolating these spectral signatures, in turn, it is possible to locate these species independent of each other in living cells of H. pluvialis in various stages of the life cycle. Chlorophyll emission was found only in the chloroplast whereas astaxanthin was identified within globular and punctate regions of the cytoplasmic space. Moreover, we found evidence for β-carotene to be co-located with both the chloroplast and astaxanthin in the cytosol. These observations imply that β-carotene is a precursor for astaxanthin and the synthesis of astaxanthin occurs outside the chloroplast. Our work demonstrates the broad utility of confocal Raman microscopy to resolve spectral signatures of highly similar chromophores in living cells.

Topics: beta Carotene; Carotenoids; Chlorophyll; Chlorophyta; Chloroplasts; Cytoplasm; Cytosol; Multivariate Analysis; Spectrum Analysis, Raman; Xanthophylls

Whole stillage--a co-product of grain-based ethanol--is used as an animal feed in the form of dried distiller's grain with solubles (DDGS). Since animals cannot synthesize carotenoids and animal feed is generally poor in carotenoids, about 30-120 ppm of total carotenoids are added to animal feed to improve animal health, enhance meat color and quality, and increase vitamin A levels in milk and meat. The main objective of this study was to produce carotenoid (astaxanthin and β-carotene)-enriched DDGS by submerged fermentation of whole stillage. Mono- and mixed cultures of red yeasts, Phaffia rhodozyma (ATCC 24202) and Sporobolomyces roseus (ATCC 28988), were used to produce astaxanthin and β-carotene. Media optimization was carried out in shake flasks using response surface methodology (RSM). Macro ingredients, namely whole stillage, corn steep liquor and glycerol, were fitted to a second-degree polynomial in RSM. Under optimized conditions, astaxanthin and β-carotene yields in mixed culture and P. rhodozyma monoculture were 5 and 278, 97, and 275 μg/g, respectively, while S. roseus produced 278 μg/g of β-carotene. Since the carotenoid yields are almost twice the quantity used in animal feed, the carotenoid-enriched DDGS has potential application as "value-added animal feed or feed blends."

Topics: Animal Feed; Animals; Basidiomycota; beta Carotene; Culture Media; Fermentation; Glycerol; Industrial Microbiology; Xanthophylls

In the screening of DNA polymerase (pol) inhibitor, we isolated lutein, a carotenoid, from the crude (unrefined) pressed oil of canola (low erucic acid rapeseed, Brassica napus L.). Commercially prepared carotenoids such as lutein (1), zeaxanthin (2), beta-cryptoxanthin (3), astaxanthin (4), canthaxanthin (5), beta-carotene (6), lycopene (7), capsanthin (8), fucoxanthin (9) and fucoxanthinol (10), were investigated for the inhibitory activities of pols. Compounds 1, 2 and 8 exhibited strong inhibition of the activities of mammalian pols beta and lambda, which are DNA repair- and/or recombination-related pols. On the other hand, all carotenoids tested had no influence on the activity of a mammalian pol alpha, which is a DNA replicative pol. Lutein (1) was the strongest pol inhibitor of mammalian pols beta and lambda in the prepared ten carotenoids tested, but did not influence of the activities of mammalian pols alpha, gamma, delta and epsilon. The tendency for pols beta and lambda inhibition by these carotenoids showed a positive correlation with the suppression of TPA (12-O-tetradecanoylphorbol-13-acetate)-induced inflammation. These results suggest that cold pressed unrefined canola/rapeseed oil, or other oils with high levels of lutein and other carotenoids, may be useful for their anti-inflammatory properties.

Topics: Animals; beta Carotene; Brassica napus; Carotenoids; Cattle; Cryptoxanthins; DNA-Directed DNA Polymerase; Enzyme Inhibitors; Humans; Lutein; Lycopene; Mammals; Molecular Structure; Nucleic Acid Synthesis Inhibitors; Rats; Xanthophylls; Zeaxanthins

Retinol (ROH) and provitamin-A carotenoids are recommended to treat ROH deficiency. Xanthophyll carotenoids, being potent antioxidants, can modulate health disorders. We hypothesize that nonprovitamin-A carotenoids may yield ROH and suppress lipid peroxidation under ROH deficiency. This study aimed to (i) study the possible bioconversion of astaxanthin and lutein to ROH similar to β-carotene and (ii) determine the antioxidant potential of these carotenoids with reference to Na(+)/K(+)-ATPase, antioxidant molecules, and lipid peroxidation (Lpx) induced by ROH deficiency in rats. ROH deficiency was induced in rats (n = 5 per group) by feeding a diet devoid of ROH. Retinol-deficient (RD) rats were gavaged with astaxanthin, lutein, β-carotene, or peanut oil alone (RD group) for 7 days. Results show that the RD group had lowered plasma ROH levels (0.3 µmol/L), whereas ROH rose in astaxanthin and β-carotene groups (4.9 and 5.7 µmol/L, respectively), which was supported by enhanced (69% and 70%) intestinal β-carotene 15,15'-monooxygenase activity. Astaxanthin, lutein, and β-carotene lowered Lpx by 45%, 41%, and 40% (plasma), respectively, and 59%, 64%, and 60% (liver), respectively, compared with the RD group. Lowered Na(+)/K(+)-ATPase and enhanced superoxide dismutase, catalase, and glutathione-S-transferase activities support the lowered Lpx. To conclude, this report confirms that astaxanthin is converted into β-carotene and ROH in ROH-deficient rats, and the antioxidant potential of carotenoids was in the order astaxanthin > lutein > β-carotene.

Topics: Animals; Antioxidants; beta Carotene; Biotransformation; Chromatography, High Pressure Liquid; Disease Models, Animal; Fatty Acids; Lipid Peroxidation; Liver; Lutein; Male; Microsomes, Liver; Rats; Rats, Wistar; Sodium-Potassium-Exchanging ATPase; Vitamin A Deficiency; Xanthophylls

Carotenoids are used for systemic photoprotection in humans. Regarding mechanisms underlying photoprotective effects of carotenoids, here we compared the modulation of UVA-related injury by carotenoids. Human dermal fibroblasts (HDF) were exposed to moderate doses of UVA, which stimulated apoptosis, increased levels of reactive oxygen species and thiobarbituric acid reactive substances, decreased antioxidant enzymes activities, promoted membrane perturbation, and induced the expression of heme oxygenase-1 (HO-1). The carotenoids astaxanthin (AX), canthaxanthin (CX) and beta-carotene (betaC) were delivered to HDF 24 h before exposure to UVA. Astaxanthin exhibited a pronounced photoprotective effect and counteracted all of the above-mentioned UVA-induced alterations to a significant extent. beta-Carotene only partially prevented the UVA-induced decline of catalase and superoxide dismutase activities, but it increased membrane damage and stimulated HO-1 expression. Moreover, betaC dose-dependently induced caspase-3 activity following UVA exposure. In contrast, CX had no effect on oxidative damage, except for HO-1 expression, which was augmented. Uptake of AX by fibroblasts was higher than that of the other two carotenoids. The photostability of the three compounds in fibroblasts was AX > CX >> betaC. The data indicate that the oxo-carotenoid AX has a superior preventive effect towards photo-oxidative changes in cell culture.

Topics: Antioxidants; Apoptosis; beta Carotene; Canthaxanthin; Caspase 3; Catalase; Cells, Cultured; Dermis; Dose-Response Relationship, Drug; Dose-Response Relationship, Radiation; Fibroblasts; Heme Oxygenase-1; Humans; Oxidative Stress; Reactive Oxygen Species; Superoxide Dismutase; Thiobarbituric Acid Reactive Substances; Ultraviolet Rays; Xanthophylls

Carotenoid biosynthesis is highly conserved and well characterized up to the synthesis of beta-carotene. Conversely, the synthesis of astaxanthin from beta-carotene is less well characterized. Regardless, astaxanthin is a highly sought natural product, due to its various industrial applications and elevated antioxidant capacity. In this article, 12 beta-carotene ketolase and 4 beta-carotene hydroxylase genes, isolated from 5 cyanobacterial species, are investigated for their function, and potential for microbial astaxanthin synthesis. Further, this in vivo comparison identifies and applies the most promising genetic elements within a dual expression vector, which is maintained in Escherichia coli. Here, combined overexpression of individual beta-carotene ketolase and beta-carotene hydroxylase genes, within a beta-carotene accumulating host, enables a 23.5-fold improvement in total carotenoid yield (1.99 mg g(-1)), over the parental strain, with >90% astaxanthin.

Topics: Bacterial Proteins; beta Carotene; Biosynthetic Pathways; Cyanobacteria; Escherichia coli; Gene Expression; Mixed Function Oxygenases; Oxygenases; Xanthophylls

The red carotenoid astaxanthin possesses higher antioxidant activity than other carotenoids and has great commercial potential for use in the aquaculture, pharmaceutical, and food industries. In this study, we produced astaxanthin in the budding yeast Saccharomyces cerevisiae by introducing the genes involved in astaxanthin biosynthesis of carotenogenic microorganisms. In particular, expression of genes of the red yeast Xanthophyllomyces dendrorhous encoding phytoene desaturase (crtI product) and bifunctional phytoene synthase/lycopene cyclase (crtYB product) resulted in the accumulation of a small amount of beta-carotene in S. cerevisiae. Overexpression of geranylgeranyl pyrophosphate (GGPP) synthase from S. cerevisiae (the BTS1 gene product) increased the intracellular beta-carotene levels due to the accelerated conversion of farnesyl pyrophosphate to GGPP. Introduction of the X. dendrorhous crtS gene, encoding astaxanthin synthase, assumed to be the cytochrome P450 enzyme, did not lead to astaxanthin production. However, coexpression of CrtS with X. dendrorhous CrtR, a cytochrome P450 reductase, resulted in the accumulation of a small amount of astaxanthin. In addition, the beta-carotene-producing yeast cells transformed by the bacterial genes crtW and crtZ, encoding beta-carotene ketolase and hydroxylase, respectively, also accumulated astaxanthin and its intermediates, echinenone, canthaxanthin, and zeaxanthin. Interestingly, we found that these ketocarotenoids conferred oxidative stress tolerance on S. cerevisiae cells. This metabolic engineering has potential for overproduction of astaxanthin and breeding of novel oxidative stress-tolerant yeast strains.

Topics: Basidiomycota; beta Carotene; Fungal Proteins; Gene Expression Regulation, Fungal; Genes, Bacterial; Genetic Engineering; Oxidative Stress; Oxygenases; Saccharomyces cerevisiae; Xanthophylls

The green microalgae Haematococcus pluvialis synthesizes secondary carotenoids after exposure to environmental stress, a process that is used for the biotechnological production of astaxanthin (Ax). This study reports, for the first time, the medium-dependent changes in the carotenoid pattern throughout the cultivation process as well as the exact composition of carotenoids and their fatty acid mono- and diesters using LC-MS. Secondary carotenoid formation started immediately upon exposure to nutrient depletion and high light conditions. Ax and its corresponding mono- and diesters were detected simultaneously. After 15 days of cultivation, no significant changes were detected in carotenoid composition; however, the ratio between carotenoid mono- and diesters still varied. Main carotenoids were identified as Ax linolenate and Ax oleate, but also five adonirubin and one lutein monoester were detected. The influence of three different autotroph media was studied on carotenoid content, which reached a maximum 16.1 mg/g dry weight. The results indicate that media composition has an influence on the ratio of Ax mono- to diester but not on the qualitative composition of secondary carotenoids in H. pluvialis. Beside the pathway via echinenone, canthaxanthin and adonirubin the results indicate that Ax biosynthesis takes place via another route: from beta-carotene via beta-cryptoxanthin, zeaxanthin and adonixanthin.

Topics: alpha-Linolenic Acid; beta Carotene; Carotenoids; Chlorophyta; Chromatography, Liquid; Cryptoxanthins; Culture Media; Lutein; Mass Spectrometry; Molecular Structure; Oleic Acid; Time Factors; Xanthophylls; Zeaxanthins

The goal of this study was to investigate the effects of beta-carotene and astaxanthin (ASX) - carotenoid without provitamin A activity on the proliferation and differentiation of rat oval cells (OC) in vitro. Oval cells were isolated from two groups of animals: I - partial hepatectomised (PH) and II- diethylnitrosamine (DEN) treated rats. At various time points cell lysates were separated by PAGE. For immunodetection primary antibodies against CD-34, Ck19 and albumin were used. Medium concentration of fibrinogen and haptoglobin was measured. Mitochondrial competence of cells was expressed as the proliferation index. In comparison to HP- and DEN-obtained oval cells cultured without carotenoids, the addition of beta-carotene and ASX increased albumin expression during the experimental period. The same condition didn't reveal CK19 expression. CD34 expressed by oval cells was detected up to the 5(th) week of beta-carotene and ASX absence in the medium. beta-carotene addition resulted in a decrease of the proliferative activity of OC, with significant changes in 48 h, the 5(th) and 15(th) week of incubation. ASX (p < or = 0.05) inhibited the proliferation, especially in 24h and 5(th) week of cell culture. In respect to haptoglobin concentration, its maximum value after the 10(th) week was observed. The fibrinogen level obtained from DEN-oval cells incubated with beta-carotene elevated from 480+/-6.87 microg/ml after 24h to 5520+/-34,56 microg/ml after the 15(th) week. In a condition without carotenoids fibrinogen concentration did not exceed 2280+/-31.5 microg/ml after the 15(th) week of cell culture.

Topics: Animals; Anticarcinogenic Agents; beta Carotene; Cell Differentiation; Cell Proliferation; Cell Transformation, Neoplastic; Diethylnitrosamine; Female; Fibrinogen; Haptoglobins; Hepatectomy; Liver; Liver Neoplasms, Experimental; Rats; Rats, Wistar; Tumor Cells, Cultured; Xanthophylls

The pathway from beta-carotene to astaxanthin is a crucial step in the synthesis of astaxanthin, a red antioxidative ketocarotenoid that confers beneficial effects on human health. Two enzymes, a beta-carotene ketolase (carotenoid 4,4'-oxygenase) and a beta-carotene hydroxylase (carotenoid 3,3'-hydroxylase), are involved in this pathway. Cyanobacteria are known to utilize the carotenoid ketolase CrtW and/or CrtO, and the carotenoid hydroxylase CrtR. Here, we compared the catalytic functions of CrtW ketolases, which originated from Gloeobacter violaceus PCC 7421, Anabaena (also known as Nostoc) sp. PCC 7120 and Nostoc punctiforme PCC 73102, and CrtR from Synechocystis sp. PCC 6803, Anabaena sp. PCC 7120 and Anabaena variabilis ATCC 29413 by complementation analysis using recombinant Escherichia coli cells that synthesized various carotenoid substrates. The results demonstrated that the CrtW proteins derived from Anabaena sp. PCC 7120 as well as N. punctiforme PCC 73102 (CrtW148) can convert not only beta-carotene but also zeaxanthin into their 4,4'-ketolated products, canthaxanthin and astaxanthin, respectively. In contrast, the Anabaena CrtR enzymes were very poor in accepting either beta-carotene or canthaxanthin as substrates. By comparison, the Synechocystis sp. PCC 6803 CrtR converted beta-carotene into zeaxanthin efficiently. We could assign the catalytic functions of the gene products involved in ketocarotenoid biosynthetic pathways in Synechocystis sp. PCC 6803, Anabaena sp. PCC 7120 and N. punctiforme PCC 73102, based on the present and previous findings. This explains why these cyanobacteria cannot produce astaxanthin and why only Synechocystis sp. PCC 6803 can produce zeaxanthin.

Topics: Bacterial Proteins; beta Carotene; Canthaxanthin; Cyanobacteria; DNA, Bacterial; Escherichia coli; Gene Expression Regulation, Bacterial; Genes, Bacterial; Genetic Complementation Test; Mixed Function Oxygenases; Oxygenases; Species Specificity; Substrate Specificity; Xanthophylls; Zeaxanthins

The antioxidant activity of beta-carotene and oxygenated carotenoids lutein, canthaxanthin, and astaxanthin was investigated during spontaneous and peroxyl-radical-induced cholesterol oxidation. Cholesterol oxidation, measured as generation of 7-keto-cholesterol (7-KC), was evaluated in a heterogeneous solution with cholesterol, AAPH, and carotenoids solubilized in tetrahydrofuran and in water, and in a homogeneous solution of chlorobenzene, with AIBN as a prooxidant. The formation of 7-KC was dependent on temperature and on cholesterol and prooxidant concentrations. All the carotenoids tested, exhibited significant antioxidant activity by inhibiting spontaneous, AAPH- and AIBN-induced formation of 7-KC, although the overall order of efficacy of these compounds was astaxanthin > canthaxanthin > lutein = beta-carotene. The finding that carotenoids exert protective effects on spontaneous and free radical-induced cholesterol oxidation may have important beneficial effects on human health, by limiting the formation of atheroma and by inhibiting cholesterol oxidation in food processing or storage.

Topics: Amidines; Antioxidants; beta Carotene; Canthaxanthin; Carotenoids; Ketocholesterols; Lutein; Oxidation-Reduction; Peroxides; Protective Agents; Solutions; Temperature; Xanthophylls

The biological benefits of certain carotenoids may be due to their potent antioxidant properties attributed to specific physico-chemical interactions with membranes. To test this hypothesis, we measured the effects of various carotenoids on rates of lipid peroxidation and correlated these findings with their membrane interactions, as determined by small angle X-ray diffraction approaches. The effects of the homochiral carotenoids (astaxanthin, zeaxanthin, lutein, beta-carotene, lycopene) on lipid hydroperoxide (LOOH) generation were evaluated in membranes enriched with polyunsaturated fatty acids. Apolar carotenoids, such as lycopene and beta-carotene, disordered the membrane bilayer and showed a potent pro-oxidant effect (>85% increase in LOOH levels) while astaxanthin preserved membrane structure and exhibited significant antioxidant activity (40% decrease in LOOH levels). These findings indicate distinct effects of carotenoids on lipid peroxidation due to membrane structure changes. These contrasting effects of carotenoids on lipid peroxidation may explain differences in their biological activity.

Topics: Antioxidants; beta Carotene; Carotenoids; Cell Membrane; Cholesterol; Fatty Acids, Unsaturated; Lipid Peroxidation; Liposomes; Lutein; Lycopene; Membrane Lipids; Molecular Conformation; Molecular Structure; X-Ray Diffraction; Xanthophylls; Zeaxanthins

Omega-3 polyunsaturated fatty acids (PUFA) are increasingly finding use as treatments for a variety of medical conditions. PUFA supplementation can, however, result in increased oxidative stress causing elevated turnover rate of membrane phospholipids, impairment of membrane integrity and increased formation of inflammatory mediators. The aim of this study was to determine which antioxidant compounds were most effective in ameliorating the stimulation of phospholipid turnover by oxidative stress. U937 cells were supplemented with eicosapentaenoic acid and either ascorbic acid, alpha-tocopherol, beta-carotene or astaxanthin prior to being challenged with oxidant. Although all antioxidants were found to be effective in decreasing oxidant-stimulated peroxide formation, only alpha-tocopherol significantly decreased oxidant-stimulated release of 3H-labeled arachidonic acid (AA), while ascorbic acid markedly increased release. All antioxidants except alpha-tocopherol decreased oxidant-stimulated 3H-AA uptake. Our data suggest that antioxidants are not equally effective in combating the effects of oxidative stress upon membrane phospholipid turnover, and that optimal protection will require mixtures of antioxidants.

Topics: alpha-Tocopherol; Antioxidants; Arachidonic Acid; Ascorbic Acid; beta Carotene; Eicosapentaenoic Acid; Humans; Lipid Peroxidation; Oxidants; Oxidative Stress; Phospholipids; U937 Cells; Xanthophylls

A simple method for the extraction of carotenoid pigments from frozen wet cells of red yeasts (Basidiomycota) and their analysis by reversed-phase HPLC using a C(18) column and a water/acetone solvent system is described. Typical red yeast carotenoids belonging to an oxidative series from the monocyclic gamma-carotene to 2-hydroxytorularhodin and from the bicyclic beta-carotene to astaxanthin were separated. Pigment identity was confirmed by LC-atmospheric pressure chemical ionisation (APCI) mass spectrometry using similar chromatographic conditions.

Topics: Basidiomycota; beta Carotene; Carotenoids; Chromatography, High Pressure Liquid; Molecular Structure; Reproducibility of Results; Xanthophylls

Carotenoids have been shown to have potential beneficial effects on human health which has led to an increasing interest in the study of their bioavailability. A Caco-2 cell model, as previously described, was employed to examine the percentage transfer of the carotenoids alpha-carotene, beta-carotene, lycopene, astaxanthin, beta-cryptoxanthin, lutein and zeaxanthin through an intact, highly differentiated Caco-2 cell monolayer at a range of different amounts. Our results show that astaxanthin, a carotenoid with powerful antioxidant capacity, had the highest percentage transfer overall. We examined the cellular uptake and secretion of lutein and zeaxanthin to compare two structurally similar carotenoids. Both were efficiently transported through the monolayer with a range between 5.1 (sem 1.2) % to 20.2 (sem 3.3) % and 5.5 (sem 2.5) % to 13.4 (sem 4) % for lutein and zeaxanthin, respectively. These carotenoids were compared to each other at each added amount and no significant difference was observed between the two xanthophylls. The carotene carotenoids alpha-carotene, beta-carotene and lycopene and the xanthophyll beta-cryptoxanthin were also examined and had lower uptake and secretion values when compared to lutein, zeaxanthin and astaxanthin. The xanthophyll beta-cryptoxanthin was also not significantly different when compared to the carotene carotenoids. Data generated from this study compares well with in vivo bioavailability studies. Furthermore, the model provides comparative data on the relative absorption and transfer of seven different carotenoids. Our data indicate that lower amounts of carotenoids were absorbed and transferred more efficiently than higher amounts suggesting a saturation effect at higher exposure.

Topics: Anticarcinogenic Agents; beta Carotene; Biological Transport; Caco-2 Cells; Carotenoids; Cryptoxanthins; Humans; Intestinal Absorption; Lutein; Lycopene; Models, Biological; Vitamins; Xanthophylls; Zeaxanthins

Most Mucor species accumulate beta-carotene as the main carotenoid. The crtW and crtZ astaxanthin biosynthesis genes from Agrobacterium aurantiacum were placed under the control of Mucor circinelloides expression signals. Expression vectors containing the bacterial genes were constructed, and PEG-mediated transformations were performed on a selected M. circinelloides strain. Transformants that exhibited altered carotene production were isolated and analyzed. Southern analysis showed that all plasmids behave as autoreplicative elements. Northern analysis detected the actual heterologous transcription products, whereas thin layer chromatography and high-performance liquid chromatography studies revealed the presence of new carotenoid compounds and intermediates among the transformants.

Topics: Bacterial Proteins; beta Carotene; Carotenoids; Chromatography, High Pressure Liquid; Chromatography, Thin Layer; Cloning, Molecular; Gene Expression; Genes, Bacterial; Genetic Vectors; Mucor; Oxygenases; Plasmids; Promoter Regions, Genetic; Recombinant Proteins; Rhizobium; RNA, Bacterial; RNA, Messenger; Transformation, Genetic; Xanthophylls

We investigated the effects of astaxanthin (AST), a carotenoid, on endotoxin-induced uveitis (EIU), and over the course of the disease measured the expression of inflammatory cytokines and chemokines in the presence or absence of AST. EIU was induced in male Lewis rats by footpad injection of lipopolysaccharide (LPS). The animals were randomly divided to 12 groups with eight animals in each. Immediately after the inoculation, AST (1, 10, or 100 mg kg(-1)) was injected intravenously. Aqueous humour was collected at 6, 12 and 24 hr after LPS inoculation and the number of infiltrating cells in the anterior chamber was counted. In addition, we assayed the concentration of protein, nitric oxide (NO), tumour necrosis factor-alpha (TNF-alpha) and prostaglandin E2 (PGE2). Immunohistochemical staining with a monoclonal antibody against activated NF-kappaB was performed in order to evaluate the effects of AST on NF-kappaB activation. Rats injected with AST showed a significant decrease in the number of infiltrating cells in the anterior chamber and additionally there was a significantly lower concentration of protein, NO, TNF-alpha and PGE2 in the aqueous humour. Moreover, even early stages of EIU were suppressed by injection of AST. The number of activated NF-kappaB-positive cells was lower in iris-ciliary bodies treated with 10 or 100 mg kg(-1) AST at 3 hr after LPS injection. These results suggest that AST reduces ocular inflammation in eyes with EIU by downregulating proinflammatory factors and by inhibiting the NF-kappaB-dependent signaling pathway.

Topics: Adjuvants, Immunologic; Animals; Aqueous Humor; beta Carotene; Ciliary Body; Depression, Chemical; Dinoprostone; Enzyme-Linked Immunosorbent Assay; Immunohistochemistry; Iris; Lipopolysaccharides; Male; NF-kappa B; Nitric Oxide; Nitrites; Random Allocation; Rats; Rats, Inbred Lew; Signal Transduction; Tumor Necrosis Factor-alpha; Uveitis; Xanthophylls

The green alga Chlorella zofingiensis produces large amounts of the valuable ketocarotenoid astaxanthin under dark, heterotrophic growth conditions, making it potentially employable for commercial production of astaxanthin as feed additives, colorants, and health products. Here, we report the identification and characterization of a beta-carotene oxygenase (CRTO) gene that is directly involved in the biosynthesis of ketocarotenoids in C. zofingiensis. The open reading frame of the crtO gene, which is interrupted by three introns of 243, 318, and 351 bp, respectively, encodes a polypeptide of 312 amino acid residues. Only one crtO gene was detected in the genome of C. zofingiensis. Furthermore, the expression of the crtO gene was transiently up-regulated upon glucose treatment. Functional complementation in Escherichia coli showed that the coding protein of the crtO gene not only exhibits normal CRTO activity by converting beta-carotene to canthaxanthin via echinenone, but also displays a high enzymatic activity of converting zeaxanthin to astaxanthin via adonixanthin. Based on the bifunctional CRTO, a predicted pathway for astaxanthin biosynthesis in C. zofingiensis is described, and the CRTO is termed as carotenoid 4,4'-beta-ionone ring oxygenase.

Topics: beta Carotene; Blotting, Southern; Chlorella; Chromatography, High Pressure Liquid; Cloning, Molecular; DNA, Complementary; Open Reading Frames; Oxygenases; Random Amplified Polymorphic DNA Technique; Reverse Transcriptase Polymerase Chain Reaction; Xanthophylls

A gene has been cloned from Xanthophyllomyces dendrorhous by complementation of astaxanthin formation in a beta-carotene accumulating mutant. It consists of 3,166 bp and contains 17 introns. For the beta-carotene mutant ATCC 96815, a single point mutation in the splicing sequence of intron 8 was found. The resulting improper splicing of the mRNA results in an inactive protein. The cDNA of this beta-carotene oxygenase encodes a cytochrome P450 monooxygenase belonging to the 3A subfamily. P450-specific domains were identified including a cytochrome P450 and an oxygen binding motif. Electrons are provided by a cytochrome P450 reductase. Functional characterization of the enzyme by genetic modification of X. dendrorhous demonstrated that this P450 monooxygenase is multifunctional catalyzing all steps from beta-carotene to astaxanthin formation by oxygenation of carbon 3 and 4. The reaction sequence is first 4-ketolation of beta-carotene followed by 3-hydroxylation. A hydroxylation mechanism at allylic carbon atoms has been proposed for the generation of 4-keto and 3-hydroxy groups at both beta-ionone ends.

Topics: Amino Acid Sequence; Base Sequence; Basidiomycota; beta Carotene; Cloning, Molecular; DNA, Complementary; DNA, Fungal; Genes, Fungal; Mixed Function Oxygenases; Models, Biological; Molecular Sequence Data; Sequence Homology, Amino Acid; Xanthophylls

In the current study, the improved oral bioavailability of a synthetic astaxanthin derivative (Cardax; disodium disuccinate astaxanthin) was utilized to evaluate its potential effects as a cardioprotective agent after 7-day subchronic oral administration as a feed supplement to Sprague-Dawley rats. Animals received one of two concentrations of Cardax in feed (0.1 and 0.4%; approximately 125 and 500 mg/kg/day, respectively) or control feed without drug for 7 days prior to the infarct study carried out on day 8. Thirty minutes of occlusion of the left anterior descending (LAD) coronary artery was followed by 2 h of reperfusion prior to sacrifice, a regimen which resulted in a mean infarct size (IS) as a percentage (%) of the area at risk (AAR; IS/AAR,%) of 61 +/- 1.8%. The AAR was quantified by Patent blue dye injection, and IS was determined by triphenyltetrazolium chloride (TTC) staining. Cardax at 0.1 and 0.4% in feed for 7 days resulted in a significant mean reduction in IS/AAR,% to 45 +/- 2.0% (26% salvage) and 39 +/- 1.5% (36% salvage), respectively. Myocardial levels of free astaxanthin achieved after 7-day supplementation at each of the two concentrations (400 +/- 65 nM and 1634 +/- 90 nM, respectively) demonstrated excellent solid-tissue target organ loading after oral supplementation. Parallel trends in reduction of plasma levels of multiple lipid peroxidation products with disodium disuccinate astaxanthin supplementation were observed, consistent with the documented in vitro antioxidant mechanism of action. These results extend the potential utility of this compound for cardioprotection to the elective human cardiovascular patient population, for which 7-day oral pre-treatment (as with statins) provides significant reductions in induced periprocedural infarct size.

Topics: Administration, Oral; Animal Feed; Animals; beta Carotene; Biological Availability; Cardiotonic Agents; Coronary Vessels; Dietary Supplements; Lipid Peroxidation; Myocardial Infarction; Myocardial Reperfusion; Oxidative Stress; Rats; Rats, Sprague-Dawley; Succinates; Xanthophylls

The conversion of beta-carotene into xanthophylls is a subject of great scientific and industrial interest. We cloned the crtS gene involved in astaxanthin biosynthesis from two astaxanthin producing strains of Xanthophyllomyces dendrorhous: VKPM Y2410, an astaxanthin overproducing strain, and the wild type ATCC 24203. In both cases, the ORF has a length of 3166 bp, including 17 introns, and codes for a protein of 62.6 kDa with similarity to cytochrome-P450 hydroxylases. crtS gene sequences from strains VKPM Y2410, ATCC 24203, ATCC 96594, and ATCC 96815 show several nucleotide changes, but none of them causes any amino acid substitution, except a G2268 insertion in the 13th exon of ATCC 96815 which causes a change in the reading frame. A G1470 --> A change in the 5' splicing region of intron 8 was also found in ATCC 96815. Both point mutations explain astaxanthin idiotrophy and beta-carotene accumulation in ATCC 96815. Mutants accumulating precursors of the astaxanthin biosynthetic pathway were selected from the parental strain VKPM Y2410 (red) showing different colors depending on the compound accumulated. Two of them were blocked in the biosynthesis of astaxanthin, M6 (orange; 1% astaxanthin, 71 times more beta-carotene) and M7 (orange; 1% astaxanthin, 58 times more beta-carotene, 135% canthaxanthin), whereas the rest produced lower levels of astaxanthin (5-66%) than the parental strain. When the crtS gene was expressed in M7, canthaxanthin accumulation disappeared and astaxanthin production was partially restored. Moreover, astaxanthin biosynthesis was restored when X. dendrorhous ATCC 96815 was transformed with the crtS gene. The crtS gene was heterologously expressed in Mucor circinelloides conferring to this fungus an improved capacity to synthesize beta-cryptoxanthin and zeaxanthin, two hydroxylated compounds from beta-carotene. These results show that the crtS gene is involved in the conversion of beta-carotene into xanthophylls, being potentially useful to engineer carotenoid pathways.

Topics: Amino Acid Substitution; Base Sequence; Basidiomycota; beta Carotene; Cloning, Molecular; Cryptoxanthins; Cytochrome P-450 Enzyme System; DNA, Fungal; Genes, Fungal; Genetic Complementation Test; Introns; Molecular Sequence Data; Molecular Weight; Mucor; Open Reading Frames; Phylogeny; Pigments, Biological; Point Mutation; Sequence Alignment; Sequence Analysis, DNA; Xanthophylls; Zeaxanthins

Astaxanthin (ASTX), a carotenoid with potent antioxidant properties, exists naturally in various plants, algae, and seafoods. In this study, we investigated the in vitro ability of ASTX to protect porcine lens crystallins from oxidative damage by iron-mediated hydroxyl radicals or by calcium ion-activated protease (calpain), in addition to the possible underlying biochemical mechanisms. ASTX (1 mM) was capable of protecting lens crystallins from being oxidized, as measured by changes in tryptophan fluorescence, in the presence of a Fenton reaction solution containing 0.2 mM Fe2+ and 2 mM H2O2. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis demonstrated that beta(high)-crystallin was the most vulnerable protein under these conditions of free radical exposure. The proteolysis of lens crystallins induced by calcium ion-activated calpain was also inhibited by ASTX (0.03-1 mM) as determined by daily measurement of the light-scattering intensity at 405 nm for five consecutive days. ASTX at 1 mM was as potent as a concentration of 0.1 mM calpain inhibitor E64 in protecting the oxidative damage/hydrolysis of porcine crystallins. At a concentration of 1 mM, ASTX provided better protection than the endogenous antioxidant glutathione in terms of suppressing calcium-induced turbidity of lens proteins. Thin-layer chromatography analysis indicated that ASTX interacted with calcium ions to form complexes, which we believe interfere with the hydrolysis of lens crystallins by calcium-activated calpain. This in vitro study shows that ASTX is capable of protecting porcine lens proteins from oxidative insults and degradation by calcium-induced calpain.

Topics: Animals; beta Carotene; Calcium; Calpain; Crystallins; Drug Interactions; Enzyme Activation; Hydroxyl Radical; Oxidation-Reduction; Oxidative Stress; Swine; Xanthophylls

Xanthophyllomyces dendrorhous (formerly Phaffia rhodozyma) in shake-flask cultures was exposed to 10-20 mmol/L H(2)O(2) at various culture stages, and the astaxanthin production was significantly increased by H(2)O(2) fed at 0 or 24 h (exponential phase), but only slightly at 48 h (near stationary phase). The astaxanthin production was enhanced most significantly with double feeding of 10 mmol/L H(2)O(2) at 0 and 24 h, reaching a cellular content of 1.30 mg/g cell and a volumetric yield of 10.4 mg/L, which were 83 and 65% higher, respectively, than those of the control (0.71 mg/g cell and 6.3 mg/L). The intracellular catalase (CAT) activity was also increased after H(2)O(2) treatment. The increases in CAT and astaxanthin of cells could be detected within 4 h of H(2)O(2) treatment. The increase in the astaxanthin content of cells was concomitant with a notable decrease in the beta-carotene content. The older yeast cells at late culture stage (120 h), due perhaps in part to their higher astaxanthin contents, were more tolerant to H(2)O(2) toxicity than the younger cells (24 h). No enhancement of the astaxanthin biosynthesis was attained when H(2)O(2) was added to the yeast culture together with a sufficient amount of exogenous CAT. The results suggest that astaxanthin biosynthesis in X. dendrorhous can be stimulated by H(2)O(2) as an antioxidative response.

Topics: Basidiomycota; beta Carotene; Catalase; DNA, Fungal; Gene Expression Regulation, Fungal; Genes, Fungal; Hydrogen Peroxide; Oxidative Stress; Xanthophylls

Solvent-induced spectral shifts of the four C40 carotenoids, beta-carotene, echinenone, canthaxantin, and astaxanthin, have been studied in supercritical CO2 and CF3H. In situ absorption spectroscopic analysis was used to determine the maximum peak position of the electronic transitions from the ground state (1(1)Ag-) to the S2 state (1(1)Bu+) of the carotenoids. The medium polarizability function, R(n) = (n2 - 1)/(n2 + 2) of the refractive index of the solvent was varied over the range R(n) = 0.08-0.14, by changing the pressure of CO2 or CF3H between 90 and 300 bar at the temperature 308 K. For all the carotenoids studied here, a significant hypsochromic shift of ca. 20-30 nm was observed in supercritical fluids as compared to that in nonpolar liquids. The spectral shifts in supercritical fluids were compared with those in liquids and showed a clear linear dependence on the medium polarizability. The temperature-dependent shift of the absorption maxima was less significant. Interestingly, there was almost no difference in the energetic position of the absorption maxima in supercritical CO2 and CF3H at a given R(n) value. This is in contrast to previous extrapolations from studies in liquids at larger R(n) values, which yielded different slopes of the R(n)-dependent spectral shifts for polar and nonpolar solvents toward the gas-phase limit of R(n) = 0. The current experimental results in the gas-to-liquid range show that the polarity of the solvent has only a minor influence on the 1(1)Ag- --> 1(1)Bu+ transition energy in the region of low R(n). We also obtain more reliable extrapolations of this 0-0 transition energy to the gas-phase limit nu(0-0)(gas-phase) approximately (23,000 +/- 120) cm(-1) for beta-carotene.

Topics: beta Carotene; Canthaxanthin; Carbon Dioxide; Carotenoids; Chemistry, Physical; Chlorofluorocarbons, Methane; Models, Chemical; Pressure; Solvents; Spectrophotometry; Temperature; Xanthophylls

Astaxanthin, a carotenoid found in plants and seafood, exhibits antiproliferative, antioxidant and anticarcinogenic properties. We show that astaxanthin delivered with tetrahydrofuran is effectively taken up by cultured colon adenocarcinoma cells and is localized mostly in the cytoplasm as detected by confocal resonance Raman and broad-band fluorescence microspectroscopy image analysis. Cells incubated with beta-carotene at the same concentration as astaxanthin (10 microM) showed about a 50-fold lower cellular amount of beta-carotene, as detected by HPLC. No detectable Raman signal of beta-carotene was found in cells, but a weak broad-band fluorescence signal of beta-carotene was observed. beta-Carotene, like astaxanthin, was localized mostly in the cytoplasm. The heterogeneity of astaxanthin and beta-carotene cellular distribution in cells of intestinal origin suggests that the possible defense against reactive molecules by carotenoids in these cells may also be heterogeneous.

Topics: beta Carotene; Chromatography, High Pressure Liquid; HT29 Cells; Humans; Spectrometry, Fluorescence; Spectrum Analysis, Raman; Xanthophylls

The green microalga Haematococcus pluvialis was cultured with different concentrations of NaNO(3) to determine the effect on cell growth and astaxanthin accumulation. The optimum nitrate concentration to obtain astaxanthin and to avoid the cessation of cell division was 0.15 g/l NaNO(3). The ratio chlorophyll a/total carotenoids proved a good physiological indicator of nitrogen deficiency in the cell. The effect of different carbon sources, malonate and acetate, on astaxanthin accumulation was also studied; up to 13 times more carotenoids per cell were accumulated in cultures with malonate than in cultures without this compound. The pigment analysis was performed by a new low toxicity HPLC method capable of separating chlorophylls a and b, carotenes and xanthophylls in a short-period of time, using low volumes of solvents and with an economical price. With this method even echinenone was separated, which had been unsuccessful by any other method.

Topics: Acetates; beta Carotene; Chlorophyll; Chlorophyll A; Chlorophyta; Chromatography, High Pressure Liquid; Dose-Response Relationship, Drug; Malonates; Nitrates; Nitrogen; Pigments, Biological; Time Factors; Xanthophylls

Astaxanthin is a natural antioxidant carotenoid that occurs in a wide variety of living organisms. We investigated, for the first time, antihypertensive effects of astaxanthin (ASX-O) in spontaneously hypertensive rats (SHR). Oral administration of ASX-O for 14 d induced a significant reduction in the arterial blood pressure (BP) in SHR but not in normotensive Wistar Kyoto (WKY) strain. The long-term administration of ASX-O (50 mg/kg) for 5 weeks in stroke prone SHR (SHR-SP) induced a significant reduction in the BP. It also delayed the incidence of stroke in the SHR-SP. To investigate the action mechanism of ASX-O, the effects on PGF(2alpha)-induced contractions of rat aorta treated with NG-nitro-L-arginine methyl ester (L-NAME) were studied in vitro. ASX-O (1 to 10 microM) induced vasorelaxation mediated by nitric oxide (NO). The results suggest that the antihypertensive effect of ASX-O may be due to a NO-related mechanism. ASX-O also showed significant neuroprotective effects in ischemic mice, presumably due to its antioxidant potential. Pretreatment of the mice with ASX-O significantly shortened the latency of escaping onto the platform in the Morris water maze learning performance test. In conclusion, these results indicate that astaxanthin can exert beneficial effects in protection against hypertension and stroke and in improving memory in vascular dementia.

Topics: Animals; Antihypertensive Agents; Aorta, Abdominal; beta Carotene; Blood Pressure; Hypertension; In Vitro Techniques; Male; Neuroprotective Agents; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Rats, Wistar; Stroke; Vasodilation; Xanthophylls

A novel method was developed for the combined determination of carotenoids and retinoids in fish eggs, which incorporates prior analyte isolation using liquid-liquid partitioning to minimize analyte degradation, and fraction analysis using high-performance liquid chromatography-electrospray (positive)-quadrupole mass spectrometry (LC-ESI(+)-MS; SIM or MRM modes). Eggs from Chinook salmon (Oncorhynchus tshawytscha) were used as the model fish egg matrix. The methodology was assessed and validated for beta-carotene, lutein, zeaxanthin, and beta-cryptoxanthin (molecular ion radicals [M](+)), canthaxanthin and astaxanthin ([M+Na](+) adducts) and all-trans-retinol ([(M+H)-H(2)O](+)). Using replicate egg samples (n=5) spiked with beta-cryptoxanthin and beta-carotene before and after extraction, matrix-sourced ESI(+) enhancement was observed as evidenced by comparable %matrix effect and %process efficiency values for beta-cryptoxanthin and beta-carotene of 114-119%. In aquaculture-raised eggs from adult Chinook salmon astaxanthin, all-trans-retinol, lutein and canthaxanthin were identified and determined at concentrations of 4.12, 1.06, 0.12 and 0.45 microg/g (egg wet weight), respectively. To our knowledge, this is the first report on a method for LC-MS determination of carotenoids and retinoids in a fish egg matrix, and the first carotenoid-specific determination in any fish egg sample.

Topics: Animals; beta Carotene; Carotenoids; Chickens; Chromatography, High Pressure Liquid; Eggs; Ovum; Salmon; Spectrometry, Mass, Electrospray Ionization; Vitamin A; Xanthophylls

To resolve the molecular basis of the coloration mechanism of alpha-crustacyanin, we used (13)C-labeled astaxanthins as chromophores for solid-state (13)C NMR and resonance Raman spectroscopy of [6,6',7,7']-(13)C(4) alpha-crustacyanin and [8,8',9,9',10,10',11,11',20,20']-(13)C(10) alpha-crustacyanin. We complement the experimental data with time-dependent density functional theory calculations on several models based on the structural information available for beta-crustacyanin. The data rule out major changes and strong polarization effects in the ground-state electron density of astaxanthin upon binding to the protein. Conformational changes in the chromophore and hydrogen-bond interactions between the astaxanthin and the protein can account only for about one-third of the total bathochromic shift in alpha-crustacyanin. The exciton coupling due to the proximity of two astaxanthin chromophores is found to be large, suggesting that aggregation effects in the protein represent the primary source of the color change.

Topics: Animals; beta Carotene; Carbon Isotopes; Carrier Proteins; Models, Molecular; Nephropidae; Nuclear Magnetic Resonance, Biomolecular; Pigments, Biological; Proteins; Quantum Theory; Spectrophotometry, Ultraviolet; Spectrum Analysis, Raman; Xanthophylls

During light induction for astaxanthin formation in Haematococcus pluvialis, we substituted photoautotrophic induction for heterotrophic induction using acetate, both to prevent contamination by heterotrophs due to addition of organic carbon and to enhance carbon assimilation in the induced cells. Strong photoautotrophic induction was performed by N-deprivation of photoautotrophically grown Haematococcus cells followed by supplementation with bicarbonate (HCO(3)(-)) or CO(2). Bicarbonate-induced cells contained more astaxanthin than acetate-induced cells, and even further enhancement of astaxanthin accumulation was achieved by continuous CO(2) supply. The maximum astaxanthin content (77.2 mg g(-1) biomass, 3.4-fold higher than with heterotrophic induction) was obtained under conditions of 5% CO(2), yielding astaxanthin concentration and productivity of 175.7 mg l(-1) and 6.25 mg l(-1) day(-1), respectively. The results indicate that photoautotrophic induction is more effective than heterotrophic induction for astaxanthin synthesis in H. pluvialis.

Topics: Acetates; beta Carotene; Bicarbonates; Carbon Dioxide; Chlorophyta; Culture Media; Light; Xanthophylls

Three keto-carotenoids were prepared by the oxidation of the stable C(40) trisulfone 6, which has been used as the key compound in our beta-carotene synthesis. The first allylic oxidation to the unsaturated ketone and the second oxidation to the alpha-hydroxyketone produced the C(40) trisulfones 7 and 10, respectively. The Ramberg-Backlund reaction of the oxidized C(40) trisulfone was efficiently effected by the use of a mild base, NaOMe, in the presence of CCl(4) as a halogenating agent to give the C(40) disulfones 8 and 11. Base-promoted dehydrosulfonation reaction of the disulfone compounds produced the fully conjugated polyenes of canthaxanthin (1), astaxanthin (2), and astacene (3).

Topics: beta Carotene; Canthaxanthin; Carotenoids; Combinatorial Chemistry Techniques; Molecular Structure; Oxidation-Reduction; Xanthophylls

Carotenoids are a naturally occurring group of compounds that possess antioxidant properties. Most natural carotenoids display poor aqueous solubility and tend to form aggregates in solution. Disodium disuccinate astaxanthin (DDA; Cardax) is a water-dispersible synthetic carotenoid that rapidly and preferentially associates with serum albumin, thereby preventing the formation of supramolecular complexes and facilitating its efficacy after parenteral administration. This study investigated the ability of DDA to reduce inflammation and myocardial injury in a rabbit model of ischemia/reperfusion. DDA (50 mg/kg/day) or saline was administered i.v. for 4 consecutive days before the initiation of the protocol for induction of myocardial ischemia/reperfusion. On the 5th day, rabbits underwent 30 min of coronary artery occlusion, followed by a 3-h reperfusion period. Myocardial infarct size, as a percentage of the area at risk, was calculated for both groups. Infarct size was 52.5 +/- 7.5% in the vehicle-treated (n = 9) and 25.8 +/- 4.7% in the DDA-treated (n = 9) animals (p < 0.01 versus vehicle; mean myocardial salvage = 51%). To evaluate the anti-inflammatory effects of DDA, complement activity was assessed at the end of reperfusion using a red blood cell lysis assay. DDA administration significantly reduced (p < 0.01) the activation of the complement system in the serum. The current results, coupled with the well established antioxidant ability of carotenoids, suggest that the mechanism(s) of action by which DDA reduces the tissue damage associated with reperfusion injury may include both antioxidant and anticomplement components.

Topics: Adjuvants, Immunologic; Animals; beta Carotene; C-Reactive Protein; Complement Activation; Complement Inactivator Proteins; Complement Membrane Attack Complex; Erythrocytes; Fluorescent Antibody Technique; Hemodynamics; Hemolysis; Male; Myocardial Infarction; Myocardial Reperfusion Injury; Myocardium; Rabbits; Tetrazolium Salts; Thiazoles; Tissue Distribution; Troponin I; Xanthophylls

Frequently used for humans as non-steroidal anti-inflammatory drug, naproxen has been known to induce ulcerative gastric lesion. The present study investigated the in vivo protective effect of astaxanthin isolated from Xanthophyllomyces dendrorhous against naproxen-induced gastric antral ulceration in rats. The oral administration of astaxanthin (1, 5, and 25 mg/kg of body weight) showed a significant protection against naproxen (80 mg/kg of body weight)-induced gastric antral ulcer and inhibited elevation of the lipid peroxide level in gastric mucosa. In addition, pretreatment of astaxanthin resulted in a significant increase in the activities of radical scavenging enzymes such as superoxide dismutase, catalase, and glutathione peroxidase. A histologic examination clearly proved that the acute gastric mucosal lesion induced by naproxen nearly disappeared after the pretreatment of astaxanthin. These results suggest that astaxanthin removes the lipid peroxides and free radicals induced by naproxen, and it may offer potential remedy of gastric ulceration.

Topics: Adjuvants, Immunologic; Animals; beta Carotene; Disease Models, Animal; Dose-Response Relationship, Drug; Glutathione Peroxidase; Male; Malondialdehyde; Naproxen; Pyloric Antrum; Rats; Rats, Sprague-Dawley; Stomach Ulcer; Superoxide Dismutase; Time Factors; Xanthophylls

Carotenoids have been implicated in numerous epidemiological studies as being protective against cancer at many sites, and their chemopreventive properties have been confirmed in laboratory studies. Astaxanthin (AST), primarily a carotenoid of marine origin, responsible for the pink coloration of salmon, shrimp and lobster, has received relatively little attention. As with other carotenoids, its highly lipophilic properties complicate delivery to model systems. To overcome this issue we have synthesized a novel tetrasodium diphosphate astaxanthin (pAST) derivative with aqueous dispersibility of 25.21 mg/ml. pAST was delivered to C3H/10T1/2 cells in an aqueous/ethanol solution and compared with non-esterified AST dissolved in tetrahydrofuran. We show pAST to (i) upregulate connexin 43 (Cx43) protein expression; (ii) increase the formation of Cx43 immunoreactive plaques; (iii) upregulate gap junctional intercellular communication (GJIC); and (iv) cause 100% inhibition of methylcholanthrene-induced neoplastic transformation at 10(-6) M. In all these assays, pAST was superior to non-esterified AST itself; in fact, pAST exceeded the potency of all other previously tested carotenoids in this model system. Cleavage of pAST to non-esterified (free) AST and uptake into cells was also verified by HPLC; however, levels of free AST were approximately 100-fold lower than in cells treated with AST itself, suggesting that pAST possesses intrinsic activity. The dual properties of water dispersibility (enabling parenteral administration in vivo) and increased potency should prove extremely useful in the future development of cancer chemopreventive agents.

Topics: Animals; Anticarcinogenic Agents; Antineoplastic Agents; Benzoates; beta Carotene; Carotenoids; Cell Line; Cell Transformation, Neoplastic; Fibroblasts; Gap Junctions; Mice; Mice, Inbred C3H; Retinoids; Xanthophylls

A derivative ratio spectrophotometric method was used for the simultaneous determination of beta-carotene and astaxanthin produced from Phaffia rhodozyma. Absorbencies of a series of the standard carotenoids in the range of 441 nm to 490 nm demonstrated that their absorptive spectra accorded with Beer's law and that the additivity when the concentrations of beta-carotene and astaxanthin and their mixture were within the range of 0 to 5 microg/ml, 0 to 6 microg/ml, and 0 to 6 microg/ml, respectively. When the wavelength interval (lambda) at 2 nm was selected to calculate the first derivative ratio spectra values, the first derivative amplitudes at 461 nm and 466 nm were suitable for quantitatively determining beta-carotene and astaxanthin, respectively. Effect of divisor on derivative ratio spectra could be neglected; any concentration used as divisor in range of 1.0 to 4.0 microg/ml is ideal for calculating the derivative ratio spectra values of the two carotenoids. Calibration graphs were established for beta-carotene within 0-6.0 microg/ml and for astaxanthin within 0-5.0 microg/ml with their corresponding regressive equations in: y=-0.0082x-0.0002 and y=0.0146x-0.0006, respectively. R-square values in excess of 0.999 indicated the good linearity of the calibration graphs. Sample recovery rates were found satisfactory (>99%) with relative standard deviations (RSD) of less than 5%. This method was successfully applied to simultaneous determination of beta-carotene and astaxanthin in the laboratory-prepared mixtures and the extract from the Phaffia rhodozyma culture.

Topics: Algorithms; Basidiomycota; beta Carotene; Spectrophotometry, Ultraviolet; Xanthophylls

Carotenoids, particularly astaxanthin, are the primary pigment in crustacean shell colour. Sub-adults of the western rock lobster, Panulirus cygnus, moult from a deep red colour (termed the red phase) to a much paler colour (the white phase) at sexual maturation. We observe a 2.4-fold difference in the amount of total carotenoid present in the shell extracts of reds compared to whites, as might be expected. However, analysis of the underlying epithelium shows that there is no correlation with shell colour and the amount of free (unesterified) astaxanthin-the level of free astaxanthin in reds and whites is not significantly different. Instead, we observe a correlated two-fold difference in the amount of esterified astaxanthin present in the epithelium of red versus white individuals. These data suggest a role for esterified astaxanthin in regulating shell colour formation and suggest that esterification may promote secretion and eventual incorporation of unesterified astaxanthin into the exoskeleton.

Topics: Animals; beta Carotene; Chromatography, High Pressure Liquid; Color; Crustacea; Epithelium; Esterification; Esters; Xanthophylls

The current study was designed to determine the effects of a dietary astaxanthin (ASX-O) on vascular reactivity in spontaneously hypertensive rats (SHR), in order to verify its antihypertensive action mechanism. We evaluated contractions induced by phenylephrine (Phe), angiotensin II (Ang II) and the xanthine/xanthine oxidase (Xan/XOD) system, and relaxations induced by sodium nitroprusside (SNP) as well as endothelium-dependent relaxations mediated by acetylcholine (ACh) in thoracic aorta of the SHR, with and without ASX-O intervention. We also investigated the effects of ASX-O on blood rheology using a microchannel array system. In this study, ASX-O showed a significant modulatory effect on nitric oxide (NO)-induced vasorelaxation by the NO-donor SNP (p<0.05). However, it did not show significant effects in restoring the impaired endothelium-dependent relaxation to ACh in the SHR. On the other hand, the constrictive effects by Phe, Ang II and Xan/XOD were ameliorated by ASX-O (p<0.05). ASX-O also demonstrated significant hemorheological effect by decreasing the microchannel transit time of whole blood. In conclusion, the results suggest that ASX-O may act in modulating the blood fluidity in hypertension, and that the antihypertensive effects of ASX-O may be exerted through mechanisms including normalization of the sensitivity of the adrenoceptor sympathetic pathway, particularly [alpha]-adrenoceptors, and by restoration of the vascular tone through attenuation of the Ang II- and reactive oxygen species (ROS)-induced vasoconstriction.

Topics: Animals; Antihypertensive Agents; Aorta; beta Carotene; Hemorheology; Hypertension; In Vitro Techniques; Male; Rats; Rats, Inbred SHR; Vasoconstriction; Vasodilation; Xanthophylls

The performance of Haematococcus pluvialis in continuous photoautotrophic culture has been analyzed, especially from the viewpoint of astaxanthin production. To this end, chemostat cultures of Haematococcus pluvialis were carried out at constant light irradiance, 1,220 microE/m2.s, and dilution rate, 0.9/d, but varying the nitrate concentration in the feed medium reaching the reactor, from 1.7 to 20.7 mM. Both growth and biomass composition were affected by the nitrate supply. With saturating nitrate, the biomass productivity was high, 1.2 g/L.d, but astaxanthin accumulation did not take place, the C/N ratio of the biomass being 5.7. Under moderate nitrate limitation, biomass productivity was decreased, as also did biomass concentration at steady state, whereas accumulation of astaxanthin developed and the C/N ratio of the biomass increased markedly. Astaxanthin accumulation took place in cells growing and dividing actively, and its extent was enhanced in response to the limitation in nitrate availability, with a recorded maximum for astaxanthin cellular level of 0.8% of dry biomass and of 5.6 mg/L.d for astaxanthin productivity. The viability of a significant continued generation of astaxanthin-rich H. pluvialis cells becomes thus demonstrated, as also does the continuous culture option as an alternative to current procedures for the production of astaxanthin using this microalga. The intensive variable controlling the behavior of the system has been identified as the specific nitrate input, and a mathematical model developed that links growth rate with both irradiance and specific nitrate input. Moreover, a second model for astaxanthin accumulation, also as a function of irradiance and specific nitrate input, was derived. The latter model takes into account that accumulation of astaxanthin is only partially linked to growth, being besides inhibited by excess nitrate. Simulations performed fit experimental data and emphasize the contention that astaxanthin can be efficiently produced under continuous mode by adjustment of the specific nitrate input, predicting even higher values for astaxanthin productivity. The developed models represent a powerful tool for management of such an astaxanthin-generating continuous process, and could allow the development of improved systems for the production of astaxanthin-rich Haematococcus cells.

Topics: beta Carotene; Biomass; Biotechnology; Cell Culture Techniques; Chlorophyta; Computer Simulation; Culture Media; Fermentation; Industrial Microbiology; Light; Nitrogen; Xanthophylls

The objectives of this study were to develop a high-performance liquid chromatography method for analysis of carotenoids in spear shrimp shells (Parapenaeopsis hardwickii) and to compare the extraction efficiency of carotenoids by supercritical carbon dioxide (SCD) and solvents. Results showed that the most appropriate HPLC method was accomplished by employing a Cosmosil 5C18-AR-II column and a mobile phase of methanol-dichloromethane-acetonitrile (90:5:5, v/v/v) (A) and water (100%) (B) with the following gradient elution: 92% A and 8% B in the beginning, decreased to 4% B in 9.5 min, 1% B in 26 min, 0% B in 35 min, maintained for 25 min, and returned to 92% A and 8% B in 61 min. All-trans-astaxanthin and its two cis isomers, as well as five astaxanthin monoesters and 11 diesters were resolved within 60 min with a flow rate at 2 mL/min and detection at 480 nm. Astaxanthin diesters were found to contain 12 fatty acids, of which palmitic acid and stearic acid constituted a large portion, whereas astaxanthin monoesters were found to contain 10 fatty acids with arachidonic acid dominating. Solvent extraction could generate a higher content of trans-astaxanthin and astaxanthin esters, while SCD extraction could produce greater levels of 9-cis-astaxanthin and 13-cis-astaxanthin.

Topics: beta Carotene; Carotenoids; Chromatography, Gas; Chromatography, High Pressure Liquid; Fatty Acids; Palmitic Acid; Penaeidae; Solvents; Stearic Acids; Xanthophylls

A simple and rapid preparative high-performance liquid chromatography (HPLC) method has been developed to isolate and characterize some minor impurities of astaxanthin using a normal-phase Lichrosorb silica column with n-hexane-acetone-tetrahydrofuran (90:2:8, v/v/v) as mobile-phase and detection at 475 nm. The isolated impurities were characterized as astacene, dehydro astacene and apoastaxanthinal by UV-vis, ESI-MS, 1H and 13C NMR spectroscopy and the molecular structures were assigned. The impurities collected using the developed conditions were over 98% pure.

Topics: beta Carotene; Chromatography, High Pressure Liquid; Magnetic Resonance Spectroscopy; Spectrometry, Mass, Electrospray Ionization; Spectrophotometry, Ultraviolet; Xanthophylls

Previous results from our laboratory have shown that a novel carotenoid derivative (disodium disuccinate astaxanthin; Cardax) produced dose-related reductions in myocardial infarct size (IS) in Sprague-Dawley rats when it was administered at any of three doses (25, 50 and 75 mg/kg, iv) on four consecutive days, followed by the acute infarct size study on day 5. Maximum salvage occurred at the highest dose (75 mg/kg) tested, and was shown as a 56% reduction in IS. In the present follow-up study, we used a more relevant large animal model, the dog, and looked at the effect of administering Cardax iv either acutely 2 h prior to occlusion (N = 8) or for 4 days at 50 mg/kg iv as previously done in the rat model (N = 6). The results were compared to a saline vehicle-treated group (N = 10). In all groups, dogs were subjected to 60 min of left anterior descending (LAD) coronary artery occlusion and 3 h of reperfusion. IS was determined using a triphenyltetrazolium chloride (TTZ) histochemical stain and was expressed as a percent of the area at risk (IS/AAR). IS/AAR was 20.9 +/- 1.6 % (mean +/- S.E.M.) in controls and was reduced to 11.0 +/- 1.7% (47.3% salvage; p < 0.01) in dogs treated only once iv at 2 h prior to occlusion, and 6.6 +/- 2.8% (68.4% salvage; p < 0.001) in dogs treated for 4 days. In the chronic treatment group, two of the three dogs with plasma concentrations of non-esterified astaxanthin above 1 microM had 0% IS/AAR (100% cardioprotection). These results suggest that Cardax has marked cardioprotective properties in both rodents and canines. Thus, Cardax may be a novel and powerful new means to prevent myocardial injury and/or necrosis associated with elective and/or urgent cardiac surgical interventions such as coronary angioplasty and stenting, as well as coronary artery bypass surgery (CABG).

Topics: Animals; beta Carotene; Cardiotonic Agents; Dogs; Heart; Myocardial Infarction; Myocardial Reperfusion; Myocardial Reperfusion Injury; Succinates; Xanthophylls

The effects of policosanol (P), of extract of red yeast rice (rice fermented with Monascus purpureus) (RYE) and of astaxanthin (A) (constituents of Armolipid) were investigated in a model of experimental atherosclerosis provoked in the rabbit by atherogenic cholesterol-enriched feed (ACEF). P and RYE and their combination were able to lower the increase of serum total cholesterol and of LDL cholesterol elicited by 3-month feeding with ACEF. They also were able to reduce the increase of blood malondialdehyde (MDA), a tracer of lipid peroxidation by the free radicals released by ACEF. When combined, the substances developed either additive or potentiated effects, supporting the rationale of their combination. Remarkable was the protective effect on lipid infiltration in the aortic wall provoked by ACEF, which was reduced by P and by RYE and almost completely prevented by the addition of A to the P-RYE combination. The results support the rationale of a combination of P, RYE and A as a useful food supplement in hyperlipemic patients.

Topics: Animals; Anticholesteremic Agents; Aorta; Arteriosclerosis; beta Carotene; Body Weight; Cholesterol; Diet, Atherogenic; Drugs, Chinese Herbal; Fatty Alcohols; Female; Free Radicals; Malondialdehyde; Monascus; Rabbits; Triglycerides; Xanthophylls

Ethanol has been found to induce ulcerative gastric lesion in humans. The present study investigated the in vivo protective effect of astaxanthin isolated from the Xanthophyllomyces dendrorhous mutant against ethanol-induced gastric mucosal injury in rats. The rats were treated with 80% ethanol for 3 d after pretreatment with two doses of astaxanthin (5 and 25 mg/kg of body weight respectively) for 3 d, while the control rats received only 80% ethanol for 3 d. The oral administration of astaxanthin (5 and 25 mg/kg of body weight) showed significant protection against ethanol-induced gastric lesion and inhibited elevation of the lipid peroxide level in gastric mucosa. In addition, pretreatment with astaxanthin resulted in a significant increase in the activities of radical scavenging enzymes such as superoxide dismutase, catalase, and glutathione peroxidase. A histologic examination clearly indicated that the acute gastric mucosal lesion induced by ethanol nearly disappeared after pretreatment with astaxanthin.

Topics: Animals; Basidiomycota; beta Carotene; Catalase; Dose-Response Relationship, Drug; Ethanol; Gastric Mucosa; Glutathione Peroxidase; Male; Malondialdehyde; Models, Biological; Mutation; Rats; Rats, Sprague-Dawley; Severity of Illness Index; Stomach Ulcer; Superoxide Dismutase; Xanthophylls

Expression of connexin 43 (Cx43) is correlated with reduced indexes of neoplasia and is upregulated by cancer-preventive retinoids and carotenoids in nontransformed human and murine fibroblasts and keratinocytes. The molecular mechanism of upregulation, however, is poorly understood. Three retinoic acid receptor (RAR) antagonists (Ro 41-5253, BMS453, and BMS493) were capable of suppressing retinoid-induced Cx43 protein expression in 10T1/2 cells. However, Ro 41-5253 did not suppress protein expression by the non-provitamin A carotenoids astaxanthin or lycopene. In contrast, Cx43 induction by astaxanthin but not by a RAR-specific retinoid was inhibited by GW9662, an antagonist of peroxisome proliferator activated receptor-gamma activation. Simultaneous treatment with the maximally effective concentration of a retinoid and with beta-carotene or the non-provitamin A carotenoid astaxanthin resulted in supraadditive upregulation of Cx43 expression, again indicating separate mechanisms of gene regulation by these two cancer preventive agents.

Topics: Animals; Anticarcinogenic Agents; Antioxidants; Benzoates; beta Carotene; Carotenoids; Cell Line; Connexin 43; Gene Expression Regulation, Neoplastic; Humans; Mice; Neoplasms; PPAR gamma; Receptors, Retinoic Acid; Retinoids; Up-Regulation; Xanthophylls

Inhibition of 5alpha-reductase has been reported to decrease the symptoms of benign prostate hyperplasia (BPH) and possibly inhibit or help treat prostate cancer. Saw Palmetto berry lipid extract (SPLE) is reported to inhibit 5alpha-reductase and decrease the clinical symptoms of BPH. Epidemiologic studies report that carotenoids such as lycopene may inhibit prostate cancer. In this investigation the effect of the carotenoid astaxanthin, and SPLE were examined for their effect on 5alpha-reductase inhibition as well as the growth of prostatic carcinoma cells in vitro. These studies support patent #6,277,417 B1. The results show astaxanthin demonstrated 98% inhibition of 5alpha-reductase at 300 microg/mL in vitro. Alphastat, the combination of astaxanthin and SPLE, showed a 20% greater inhibition of 5alpha-reductase than SPLE alone n vitro. A nine day treatment of prostatic carcinoma cells with astaxanthin in vitro produced a 24% decrease in growth at 0.1 mcg/mL and a 38% decrease at 0.01 mcg/mL. SPLE showed a 34% decrease at 0.1 mcg/mL.. Low levels of carotenoid astaxanthin inhibit 5alpha-reductase and decrease the growth of human prostatic cancer cells in vitro. Astaxanthin added to SPLE shows greater inhibition of 5alpha-reductase than SPLE alone in vitro.

Topics: 3-Oxo-5-alpha-Steroid 4-Dehydrogenase; Androgen Antagonists; beta Carotene; Cell Line, Tumor; Dose-Response Relationship, Drug; Gene Expression Regulation, Neoplastic; Humans; In Vitro Techniques; Male; Plant Extracts; Prostatic Hyperplasia; Prostatic Neoplasms; Serenoa; Xanthophylls

Astaxanthin (Asx), one of the carotenoids, is a red pigment in fish and Crustaceans, and possesses stronger reduction properties than conventional carotenoids, like beta-carotene. However, little is known about the biochemical properties and physiological functions of astaxanthin. The effects of astaxanthin and vitamin C on stressed rats were studied physiologically and biochemically. beta-Carotene and three kinds of astaxanthins, which were extracted from Haematococcus and Phaffia, and synthesized chemically, were used in these experiments. These rats given astaxanthins or beta-carotene had stress induced on the 12th day by immersing the rats in chest-level water at 20 degrees C for 24 h after fasting for 24 h. Rats given astaxanthins or beta-carotene prior to stressing were appreciably protected against the evolution of gastric ulcerations in relation to control rats. Ulcer indexes in particular were smaller with the rat group fed astaxanthin extracted from Haematococcus than the other groups. Next, the effects of Asx and/or vitamin C on the protection of evolution of gastric ulcer in stressed rats were persued by the same methods as described above. The results showed that rats given Asx or vitamin C were appreciably protected against the evolution of gastric ulcerations in relation to control rats. The effects were more intense, especially in rats simultaneously supplied Asx and vitamin C than in rats taking either Asx or vitamin C. It was suggested that the simultaneous supplementation of food substances with astaxanthin and vitamin C would supply enough antioxidants to offset stress-related injuries.

Topics: Alanine Transaminase; Animals; Antioxidants; Ascorbic Acid; Aspartate Aminotransferases; beta Carotene; Diet; gamma-Glutamyltransferase; Immersion; Male; Rats; Rats, Wistar; Stomach Ulcer; Stress, Physiological; Triglycerides; Xanthophylls

The ultrafast internal conversion (IC) dynamics of seven C(40) carotenoids have been investigated at room temperature in a variety of solvents using two-color transient lens (TL) pump-probe spectroscopy. We provide comprehensive data sets for the carbonyl carotenoids canthaxanthin, astaxanthin, and-for the first time-echinenone, as well as new data for lycopene, beta-carotene, (3R,3'R)-zeaxanthin and (3R,3'R,6'R)-lutein in solvents which have not yet been investigated in the literature. Measurements were carried out to determine, how the IC processes are influenced by the conjugation length of the carotenoids, additional substituents and the polarity of the solvent. TL signals were recorded at 800 nm following excitation into the high energy edge of the carotenoid S2 band at 400 nm. For the S2 lifetime solvent-independent upper limits on the order of 100-200 fs are estimated for all carotenoids studied. The S1 lifetimes are in the picosecond range and increase systematically with decreasing conjugation length. For instance, in the sequence canthaxanthin/echinenone/beta-carotene (13/12/11 double bonds) one finds tau1 approximately 5, 7.7 and 9 ps for the S1-->S0 IC process, respectively. Hydroxyl groups not attached to the conjugated system have no apparent influence on tau1, as observed for canthaxanthin/astaxanthin (tau1 approximately 5 ps in both cases). For all carotenoids studied, tau1 is found to be insensitive to the solvent polarity. This is particularly interesting in the case of echinenone, canthaxanthin and astaxanthin, because earlier measurements for other carbonyl carotenoids like, e.g., peridinin partly showed dramatic differences. The likely presence of an intramolecular charge transfer state in the excited state manifold of C40 carbonyl carotenoids, which is stabilized in polar solvents, has obviously no influence on the measured tau1.

Topics: Antioxidants; beta Carotene; Canthaxanthin; Carotenoids; Lenses; Lutein; Lycopene; Microscopy, Confocal; Solvents; Time Factors; Xanthophylls; Zeaxanthins

Medium composition was optimized for high-level production of astaxanthin by Xanthophyllomyces dendrorhous mutant JH1 using statistical experimental designs. Glucose and yeast extract were the most important factors affecting astaxanthin production. Glucose 3.89%, yeast extract 0.29%, KH2PO4 0.25%, MgSO4 0.05%, MnSO4 0.02%, and CaCl2 0.01% were optimum for high-level production of astaxanthin. Under optimized conditions, the maximum concentration of astaxanthin obtained after 7 d of cultivation was 36.06 mg/l. The concentration of astaxanthin predicted by a polynomial model was 36.16 mg/l.

Topics: Basidiomycota; beta Carotene; Culture Media; Data Interpretation, Statistical; Glucose; Models, Statistical; Mutation; Research Design; Xanthophylls; Yeasts

We assessed the effects of dietary carotenoid pigment supplementation on liver histochemistry in the rainbow trout. One hundred and eight rainbow trout (mean mass 266+/-10 g) were assigned to each of three replicate tanks for each of three dietary treatments; astaxanthin, canthaxanthin, or control at a target dietary inclusion of 100 mg/kg, by top-coating a pigment-free commercially extruded basal diet (Trouw Aquaculture, U.K.). Fish were fed for 3 weeks at a ration of 1.2% body mass/day, in a recirculating freshwater system maintained at 16 degrees C. Frozen liver sections were stained for total lipids, unsaturated lipids, glycogen, mucopolysaccharides, glycogen phosphorylase and aspartate aminotransferase. Relative amounts were measured quantitatively by image analysis. Carotenoid treatment significantly (P<0.05) altered the total lipid profile and hepatic mucopolysaccharide contents of livers of rainbow trout. Results are discussed in relation to the catabolic potential of the liver in carotenoid pigment metabolism.

Topics: Animals; beta Carotene; Canthaxanthin; Carotenoids; Dietary Supplements; Glycosaminoglycans; Lipid Metabolism; Lipids; Liver; Oncorhynchus mykiss; Pigments, Biological; Xanthophylls

Astaxanthin has been shown to have antiproliferative activity on breast cancer and skin cancer cells. However, the high cost of production, isolation and purification of purified astaxanthin from natural sources or chemically synthetic methods limit its usage on cancer therapy. We show that astaxanthin could be produced by fermentating the Phaffia rhodozyma (Xanthophyllomyces dendrorhous) yeast cells with brewer malt waste using a 20 L B. Braun fermentor. The percentage composition of astaxanthin from the P. rhodozyma was >70% of total pigment as estimated by the high performance liquid chromatographic analysis. Furthermore, the antiproliferative activity of this P. rhodozyma cell extract (PRE) was demonstrated on breast cancer cell lines including the MCF-7 (estrogen receptor positive) and MDA-MB231 (estrogen receptor negative) by using the [3-(4,5-dimethylthiazol-2-yl)-5-(3-arboxymethoxyphenyl)-2- (4-sulfophenyl)-2H-tetrazolium] (MTS) assay. No apoptotic cell death, but growth inhibitory effect was induced after 48 h of PRE incubation as suggested by morphological investigation. Anchorage-dependent clonogenicity assay showed that PRE could reduce the colony formation potential of both breast cancer cell lines. Cell death was observed from both breast cancer cell lines after incubation with PRE for 6 days. Taken together, our results showed that by using an economic method of brewer malt waste fermentation, we obtained P. rhodozyma with a high yield of astaxanthin and the corresponding PRE could have short-term growth inhibition and long-term cell death activity on breast cancer cells.

Topics: Antibiotics, Antineoplastic; Apoptosis; Basidiomycota; beta Carotene; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; DNA Fragmentation; Edible Grain; Female; Fermentation; Humans; Waste Products; Xanthophylls

The effects of feed intake, growth rate and temperature (8 and 12 degrees C) on apparent digestibility coefficients (ADC), blood uptake of individual astaxanthin E/Z isomers and metabolism of astaxanthin (3,3'-dihydroxy-beta,beta-carotene-4,4'-dione) were determined in Atlantic salmon. Accumulation of idoxanthin (3,4,3'-trihydroxy-beta,beta-carotene-4-one) in plasma was used to indicate metabolic transformation of astaxanthin. Quadruplicate groups of fish were subjected to three different treatments; one treatment was kept at 12 degrees C and fed to satiation. Another treatment kept at 12 degrees C was pair-fed with fish fed to satiation at 8 degrees C, resulting in a restricted feeding regime for the former treatment. After 2 months of feeding, the fish were fed a single meal containing ballotini glass beads to determine individual feed intake and Y(2)O(3) as an inert marker to determine ADCs. The faeces samples were pooled into 6 categories according to individual meal size (range 0.2-1.5% of body weight) and the ADCs for different meal sizes were determined. ADCs of astaxanthin ranged from 20% to 60% but were not significantly correlated with meal size. However, fish kept at 12 degrees C had approximately 10% higher ADC than fish kept at 8 degrees C (p=0.032). Growth rate and plasma astaxanthin concentration were higher at higher temperature and higher ration. Plasma concentration of idoxanthin was not affected by temperature or by meal size. The incidence of fin erosion and non-feeding individuals was significantly higher among fish fed a restricted ration indicating more aggressive interactions. Fish with visible fin damage had a tendency for having higher idoxanthin content in plasma than fish without noticeable fin damage. It is concluded that temperature but not individual meal size affect ADC of astaxanthin, whereas both influence plasma astaxanthin levels and may therefore affect the efficiency of astaxanthin utilization.

Topics: Animals; Atlantic Ocean; beta Carotene; Carotenoids; Digestion; Eating; Salmo salar; Social Behavior; Temperature; Xanthophylls

To increase the cell concentration and the accumulation of astaxanthin, the effects of the fed-batch addition of 10-fold-concentrated medium to supply nutrients, as well as illumination with blue light emitting diodes (LEDs), on cell growth and accumulation of astaxanthin were studied for the cultivation of Haematococcus pluvialis. Using the fed-batch addition method, the cell concentration increased above 1 mg-dry cell/cm3, and under illumination with blue LEDs, the astaxanthin concentration reached approximately 70 microg/cm3. This method was much simpler to operate than the medium replacement method in operation and enabled us to attain a higher total yield of astaxanthin.

Topics: beta Carotene; Biotechnology; Cell Culture Techniques; Chlorophyta; Light; Xanthophylls

Astaxanthin is a carotenoid found in plants and algae; it provides the color of marine seafood such as salmon, lobster, or shrimp. Carotenoids are antioxidants and exhibit other biological functions, including effects on gap junctional communication important for homeostasis, growth control, and development of cells. Cancer cells have an impaired gap junctional intercellular communication. The objective of the present study was to determine the effects of astaxanthin and canthaxanthin on gap junctional intercellular communication in vitro. Primary human skin fibroblasts were exposed to carotenoids from 0.001 to 10 micromol/L, and gap junctional communication was measured with a dye transfer assay. After incubation with canthaxanthin for 24 and 72 h, intercellular communication increased, whereas it was strongly diminished by astaxanthin at levels > 0.1 micromol/L. Inhibition was reversed when astaxanthin was withdrawn. Western blot analysis showed that after exposure to canthaxanthin, the amount of the gap junction protein connexin43 was increased. Incubation with astaxanthin led to a change in the phosphorylation pattern of connexin43, shifting from higher to lower phosphorylation states. We suggest that astaxanthin affects channel function by changing the phosphorylation pattern of connexin43.

Topics: Anticarcinogenic Agents; beta Carotene; Cell Communication; Cell Membrane; Cells, Cultured; Connexin 43; Fibroblasts; Gap Junctions; Humans; Immunohistochemistry; Phosphorylation; Xanthophylls

Carotenoids are unstable and susceptible to disruption by environmental factors such as heat, light, and solvents. However, there is little information on the effect of metal ions on stability of carotenoids, especially those essential elements in human nutrition. Astaxanthin is one of the few carotenoids containing four oxygen donors. Usually, these oxygen donors can coordinate with heavy metal ions such as Cu(II) and Fe(III). In the present study, the interaction of trans-astaxanthin with Cu(II) was examined. It was found that Cu(II) markedly induces the conversion of trans-astaxanthin to its cis forms, which mainly consist of 9-cis-astaxanthin and 13-cis-astaxanthin as suggested by UV-visible spectra and HPLC measurements. Increasing either incubation time of Cu(II) and trans-astaxanthin in ethanol or the Cu(II)/astaxanthin ratio results in an increased percentage of cis isomers derived from trans-astaxanthin. All these results provide important information on the effects of dietary factors on the bioavailability and bioactivity of trans-astaxanthin.

Topics: beta Carotene; Chromatography, High Pressure Liquid; Copper; Ethanol; Isomerism; Spectrophotometry; Xanthophylls

The paper presents basic data on astaxanthin adsorption from fisheries wastewater to fish scales. This process has been proposed to be applicable in fisheries and shrimp waste management [Helgason, Recovery of compounds using a natural adsorbent, Patent WO 01/77230, 2001]. The innovative feature of the method is the application of a solid waste (fish scales) as a natural adsorbent for a carotenoid pigment (astaxanthin) from the seafood industry wastewater. The model investigations were performed with pure synthetic carotenoids to exclude the role of matrix in which astaxanthin is present in the wastewater. Under the experimental conditions used, the maximum loading capacity of astaxanthin onto the scales is 360 mg kg(-1) dry wt. Studies of the thus formed value added product indicated that drying causes significant loss of astaxanthin activity. Due to the effective filtration characteristics of the studied sorption material, we suggest the scale/astaxanthin sorption process to be suitable for treatment of wastewater from different industries.

Topics: Adsorption; Animal Structures; Animals; beta Carotene; Fishes; Industrial Waste; Kinetics; Particle Size; Water Pollutants, Chemical; Water Purification; Xanthophylls

When grown photoautotrophically, Chlorella zofingiensis strain CCAP 211/14 accumulates a significant amount of valuable carotenoids, namely astaxanthin and lutein, of increasing demand for use as feed additives in fish and poultry farming, as colorants in food, and in health care products. Under standard batch-culture conditions, this microalgal strain exhibits high values of both growth rate (about 0.04 h(-1)) and standing cell population (over 10(11) cells l(-1), or 7 g dry weight l(-1)). Lutein, in a free (unesterified) form, was the prevalent carotenoid during early stages of cultivation (over 0.3 pg cell(-1), equal to 4 mg g(-1) dry weight, or 20 mg l(-1) culture), whereas esterified astaxanthin accumulated progressively, to reach a maximum (over 0.1 pg cell(-1), equal to 1.5 mg g(-1) dry weight, or 15 mg l(-1) culture) in the late stationary phase. A differential response of lutein and astaxanthin accumulation was also recorded with regard to the action of some environmental and nutritional factors. C. zofingiensis CCAP 211/14 represents a unique model system for analyzing the differential regulation of the levels of primary (lutein) and secondary (astaxanthin) carotenoids. Relevant also from the biotechnological viewpoint, this photosynthetic organism, with outstanding attributes for fast photosynthetic growth and carotenoid accumulation, might prove most valuable for its application to the mass production of either or both lutein and astaxanthin.

Topics: beta Carotene; Chlorella; Culture Media; Light; Lutein; Nitrates; Nutritional Requirements; Sodium Chloride; Temperature; Xanthophylls

Astaxanthin extracted from green algae is desirable in the food and pharmaceutical industries due to its antioxidant properties. The green unicellular clear water microalga Haematococcus pluvialis has a high production rate of astaxanthin; indeed, it contains more than 80% astaxanthin content in its cells. This remarkable astaxanthin production is commonly obtained under stress conditions such as nutrient deficiency (N or P), high NaCl concentrations, variations of temperature, and other factors. In this vein, a great research effort has been oriented to determine optimal conditions for astaxanthin production by H. pluvialis. The objective of the present study was the analysis of environmental factors, such as light intensity, aeration and nutrients on the growth and astaxanthin production of H. pluvialis. Maximum growth of H. pluvialis obtained was 3.5x10(5) cells/ml in BBM medium at 28 degrees C under continuous illumination (177 micromol photon m(-2)s(-1)) of white fluorescent light, with continuous aeration (1.5 v.v.m.). Meanwhile, maximal astaxanthin production was 98 mg/g biomass in BAR medium with continuous illumination (345 micromol photon m(-2)s(-1)), with 1 g/l of sodium acetate and without aeration.

Topics: Air; Analysis of Variance; Antioxidants; beta Carotene; Chlorophyta; Environment; Light; Nutritional Physiological Phenomena; Sodium Acetate; Time Factors; Xanthophylls

A novel role of ovorubin as a protection system against oxidative damage in eggs from Pomacea canaliculata was investigated. Carotenoid composition, and their antioxidant capacity, as well as the carotenoid-apoprotein interaction, were studied for this lipoglycocarotenoprotein. Carotenoid extracts from ovorubin were analysed by TLC and spectrophotometry. The major carotenoid was astaxanthin in its free (40%), monoester (24%), and diester (35%) forms, mainly esterified with 16:0 fatty acid. The antioxidant capacity of ovorubin carotenoids was studied by the inhibition of microsomal oxidation in a non-enzymatic system, showing strong protection against oxidative damage (IC50=3.9 nmol/mg protein). The carotenoid-apoprotein interaction was studied by spectrophotometry and electrophoresis using reconstituted ovorubin. Astaxanthin does not seem to affect the structural characteristics of ovorubin, however the carotenoid-protein association significantly protected astaxanthin against oxidation. Ovorubin therefore, besides its role in providing energy and structural precursors during embryogenesis, would be an antioxidant carrier, protecting at the same time this pigment from oxidation in the perivitellin fluid environment of the egg.

Topics: Animals; Antioxidants; Apoproteins; beta Carotene; Carotenoids; Dose-Response Relationship, Drug; Egg Proteins; Fatty Acids; Female; Fluorescence; Microsomes, Liver; Oxidation-Reduction; Rats; Snails; Xanthophylls

A fungal contaminant on an agar plate containing colonies of Xanthophyllomyces dendrorhous markedly increased carotenoid production by yeast colonies near to the fungal growth. Spent-culture filtrate from growth of the fungus in yeast-malt medium also stimulated carotenoid production by X. dendrorhous. Four X. dendrorhous strains including the wild-type UCD 67-385 (ATCC 24230), AF-1 (albino mutant, ATCC 96816), Yan-1 (beta-carotene mutant, ATCC 96815) and CAX (astaxanthin overproducer mutant) exposed to fungal concentrate extract enhanced astaxanthin up to approximately 40% per unit dry cell weight in the wild-type strain and in CAX. Interestingly, the fungal extract restored astaxanthin biosynthesis in non-astaxanthin-producing mutants previously isolated in our laboratory, including the albino and the beta-carotene mutant. The fungus was identified as Epicoccum nigrum by morphology of sporulating cultures, and the identity confirmed by genetic characterization including rDNA sequencing analysis of the large-subunit (LSU), the internal transcribed spacer, and the D1/D2 region of the LSU. These E. nigrum rDNA sequences were deposited in GenBank under accesssion numbers AF338443, AY093413 and AY093414. Systematic rDNA homology alignments were performed to identify fungi related to E. nigrum. Stimulation of carotenogenesis by E. nigrum and potentially other fungi could provide a novel method to enhance astaxanthin formation in industrial fermentations of X. dendrorhous and Phaffia rhodozyma.

Topics: Base Sequence; Basidiomycota; beta Carotene; Carotenoids; DNA Primers; DNA, Fungal; Mitosporic Fungi; Phylogeny; Polymerase Chain Reaction; Xanthophylls

Curcumin manganese complex (CpCpx) and diacetylcurcumin manganese complex (AcylCpCpx) were determined as to their effect on the nitric oxide (NO) radical scavenging in vitro method using a sodium nitroprusside generating NO system compared with their parent compound and astaxanthin, an extreme antioxidant. All compounds effectively reduced the generation of NO radicals in a dose dependent manner. They exhibited strong NO radical scavenging activity with low IC(50) values. The IC(50) values of curcumin, diacetylcurcumin, CpCpx and AcylCpCpx obtained are 20.39+/-4.10 microM, 28.76+/-1.48 microM, 9.79+/-1.50 microM and 8.09+/-0.99 microM, respectively. CpCpx and AcylCpCpx show greater NO radical scavenging than their parent compounds, curcumin and acetylcurcumin, respectively. However, the IC(50) values of curcumin and related compounds were found to be less than astaxanthin, an extreme antioxidant, with the lower IC(50) value of 3.42+/-0.50 microM.

Topics: beta Carotene; Curcumin; Free Radical Scavengers; Manganese; Nitric Oxide; Nitroprusside; Quercetin; Structure-Activity Relationship; Xanthophylls

This study was undertaken to identify novel approaches to pharmacological treatment of asthma. Here we hypothesize that the platelet-activating factor receptor antagonist ginkgolide B (GB) in combination with the antioxidant carotenoid astaxanthin (ASX) suppresses T cell activation comparably to two commonly-used antihistamines: cetirizine dihydrochloride (CTZ) and azelastine (AZE). Peripheral blood mononuclear cells from asthmatics, cultured 24 h with either 50 microg/ml phytohemaglutinin (PHA) or PHA plus selected dosages of each drug are analyzed by flow cytometry for CD25+ or HLA-DR+ on CD3+ (T cells). Results are reported as stimulation indices (SI) of %CD3+CD25+ cells or %CD3+HLA-DR+ cells in cultures treated with PHA alone versus these subpopulations in cultures treated with both PHA and drugs. Combinations of ASX and GB exhibited optimal suppression at 10(-7) M GB + 10(-8) M ASX for CD3+CD25+ (SI = 0.79 +/- 0.04, P = 0.001) and 10(-7) M GB + 10(-7) M ASX for CD3+HLA-DR+ (SI = 0.82 +/- 0.05, P = 0.004). In conclusion, suppression of T cell activation below fully stimulated values by GB, ASX, and their combinations was comparable and for some combinations better than that mediated by CTZ and AZE. These results suggest that ASX and GB may have application as novel antiasthmatic formulations.

Topics: Adjuvants, Immunologic; Adult; Asthma; beta Carotene; Cells, Cultured; Diterpenes; Drug Combinations; Female; Ginkgolides; Humans; Lactones; Leukocytes, Mononuclear; Lymphocyte Activation; Male; Plant Extracts; Platelet Membrane Glycoproteins; Receptors, G-Protein-Coupled; T-Lymphocytes; Xanthophylls

The green microalga Haematococcus pluvialis can synthesize and accumulate large amounts of the ketocarotenoid astaxanthin, and undergo profound changes in cell wall composition and architecture during the cell cycle and in response to environmental stresses. In this study, cell wall proteins (CWPs) of H. pluvialis were systematically analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) coupled with peptide mass fingerprinting (PMF) and sequence-database analysis. In total, 163 protein bands were analyzed, which resulted in positive identification of 81 protein orthologues. The highly complex and dynamic composition of CWPs is manifested by the fact that the majority of identified CWPs are differentially expressed at specific stages of the cell cycle along with a number of common wall-associated 'housekeeping' proteins. The detection of cellulose synthase orthologue in the vegetative cells suggested that the biosynthesis of cellulose occurred during primary wall formation, in contrast to earlier observations that cellulose was exclusively present in the secondary wall of the organism. A transient accumulation of a putative cytokinin oxidase at the early stage of encystment pointed to a possible role in cytokinin degradation while facilitating secondary wall formation and/or assisting in cell expansion. This work represents the first attempt to use a proteomic approach to investigate CWPs of microalgae. The reference protein map constructed and the specific protein markers obtained from this study provide a framework for future characterization of the expression and physiological functions of the proteins involved in the biogenesis and modifications in the cell wall of Haematococcus and related organisms.

Topics: beta Carotene; Cell Cycle; Cell Division; Cell Wall; Chlorophyta; Chromatography, High Pressure Liquid; Databases as Topic; Electrophoresis, Polyacrylamide Gel; Flagella; Flow Cytometry; Glucosyltransferases; Heat-Shock Proteins; Hydrolysis; Mass Spectrometry; Microscopy; Microscopy, Electron; Oxidation-Reduction; Oxidoreductases; Peptides; Protein Binding; Proteomics; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Time Factors; Xanthophylls

The present studies were performed to investigate the metabolism of astaxanthin (Ax) in Atlantic salmon, especially in the liver of salmon. The investigations were undertaken in vivo salmon that were fed a diet containing 60 ppm 15, 15' 14C-labelled Ax prior to sacrifice. The samples of blood, bile, liver, gastrointestinal tract and contents, muscle, skin, remaining carcass and feces were taken for scintillation counting. The highest radioactivity (71.36%) of 14C-labelled Ax was found in the gastrointestinal contents and feces, 7.13% in the bile and 10.68% in the samples of liver, muscle, and skin at the end of the experiments. The metabolites of 14C-labelled Ax were extracted from the bile of the salmon and analyzed using thin-layer chromatography (TLC) and high performance liquid chromatography (HPLC). Predominant 14C-labelled Ax and its cis-isomers were found and no conjugation of 14C-labelled Ax was observed. These results indicate that 14C-labelled Ax was not conjugated into larger colorless compound in Atlantic salmon liver.

Topics: Animals; Aquaculture; beta Carotene; Bile; Carbon Radioisotopes; Chromatography, High Pressure Liquid; Chromatography, Thin Layer; Diet; Feces; Liver; Salmo salar; Scintillation Counting; Xanthophylls

The aim of this study was to evaluate the influence of -tocopherol and astaxanthin on low-density lipoprotein (LDL) oxidation lag time and atherosclerotic lesion formation in Watanabe heritable hyperlipidemic (WHHL) rabbits. Thirty-one, 3-month-old WHHL rabbits were divided into three experimental groups. One group (n=10) was fed standard rabbit feed alone and served as a control, a second group (n=11) was supplied with the same feed containing 500 mg alpha-tocopherol/kg and a third group (n=10) was given a feed containing 100 mg astaxanthin/kg. Plasma lipids, lipoproteins and LDL oxidation lag time were followed for 24 weeks. At the end of the treatment period, the animals were killed and the thoracic aorta was used for evaluation of the degree of atherosclerosis. Colour photographs of the intimal surface of the vessel were taken for determination of the atherosclerotic area. Cross-sections of the thoracic aorta were used for histological examination and for determination of intimal thickening. Specimens of the vessel were used for determination of the tissue cholesterol content. Plasma cholesterol remained at a high level during the time of the experiment and there were no differences between the experimental groups. After 24 weeks, the LDL oxidation lag time was 53.7+/-1.7 min, 109+/-4 min (P<0.001) and 56.4+/-3.4 min (P=0.47) in the control, alpha-tocopherol and astaxanthin groups, respectively. In the thoracic aorta, the atherosclerotic area was 80.7+/-5.1%, 67.1+/-6.7% (P=0.13) and 75.2+/-5.7% (P=0.49) in the control, alpha-tocopherol and astaxanthin groups, respectively. The intimal thickening was 45.6+/-3.2%, 44.0+/-4.1% (P=0.89) and 40.0+/-4.5% (P=0.33) in the control, alpha-tocopherol and astaxanthin groups, respectively. Finally, the cholesterol content was 107+/-9 mol/g, 95.7+/-11.5 mol/g (P=0.31) and 101+/-5 mol/g (P=0.33) in the control, alpha-tocopherol and astaxanthin groups, respectively. It can be concluded that alpha-tocopherol but not astaxanthin prolonged the LDL oxidation lag time. The two antioxidative substances did not prevent atherogenesis in WHHL rabbits in this setting.

Topics: alpha-Tocopherol; Animals; Arteriosclerosis; beta Carotene; Biopsy, Needle; Disease Models, Animal; Female; Hyperlipoproteinemia Type II; Immunohistochemistry; Lipid Peroxidation; Lipoproteins, LDL; Male; Probability; Rabbits; Reference Values; Sensitivity and Specificity; Xanthophylls

In this work, molecularly imprinted microspheres (MIMs) were synthesized by aqueous microsuspension polymerization using astaxanthin (3,3'-dihydroxy-beta,beta'-carotene-4,4'-dione) as imprinting molecule. The MIMs obtained were subsequently packed into the stainless steel column and the chromatographic characterization of the column was investigated. The effects of pH and composition of the mobile phase on the retention factor (k') were investigated in detail. The mixture of methanol and dichloromethane (DCM) (8:2, v/v) was used as mobile phase A while the mixture of methanol and water (5:5, v/v) as mobile phase B. The separation of astaxanthin and zeaxanthin (3,3'-dihydroxyl-beta-carotene) was obtained when the concentration of mobile phase B was higher than 30% (v/v) due to their strong lipophilicity. The method developed was successfully applied to separate astaxanthin in the saponified samples of the microalga Haematococcus pluvialis and the yeast Phaffia rhodozyma. The recovery of adding 40 mg astaxanthin to 1.0 g microalgal sample was 95.5% with an R.S.D. (n =5) of 5.3%. The results of determination of astaxanthin in the microalga and the yeast were 3.7% (R.S.D (n = 1.5%, n = 9) and 0.041% (R.S.D n= 7.3%, n = 9), respectively.

Topics: beta Carotene; Eukaryota; Hydrogen-Ion Concentration; Microspheres; Xanthophylls; Yeasts

Oxidative stress is implicated as an important mechanism by which diabetes causes nephropathy. Astaxanthin, which is found as a common pigment in algae, fish, and birds, is a carotenoid with significant potential for antioxidative activity. In this study, we examined whether chronic administration of astaxanthin could prevent the progression of diabetic nephropathy induced by oxidative stress in mice. We used female db/db mice, a rodent model of type 2 diabetes, and their non-diabetic db/m littermates. The mice were divided into three groups as follows: non-diabetic db/m, diabetic db/db, and diabetic db/db treated with astaxanthin. Blood glucose level, body weight, urinary albumin, and urinary 8-hydroxydeoxyguanosine (8-OHdG) were measured during the experiments. Histological and 8-OHdG immunohistochemical studies were performed for 12 weeks from the beginning of treatment. After 12 weeks of treatment, the astaxanthin-treated group showed a lower level of blood glucose compared with the non-treated db/db group; however, both groups had a significantly high level compared with the db/m mice. The relative mesangial area calculated by the mesangial area/total glomerular area ratio was significantly ameliorated in the astaxanthin-treated group compared with the non-treated db/db group. The increases in urinary albumin and 8-OHdG at 12 weeks of treatment were significantly inhibited by chronic treatment with astaxanthin. The 8-OHdG immunoreactive cells in glomeruli of non-treated db/db mice were more numerous than in the astaxanthin-treated db/db mice. In this study, treatment with astaxanthin ameliorated the progression and acceleration of diabetic nephropathy in the rodent model of type 2 diabetes. The results suggested that the antioxidative activity of astaxanthin reduced the oxidative stress on the kidneys and prevented renal cell damage. In conclusion, administration of astaxanthin might be a novel approach for the prevention of diabetes nephropathy.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Albuminuria; Animals; beta Carotene; Deoxyguanosine; Diabetes Mellitus, Type 2; Diabetic Nephropathies; Disease Models, Animal; Female; Kidney; Mice; Mice, Mutant Strains; Reference Values; Regression Analysis; Tissue Distribution; Xanthophylls

Raman spectroscopy (785 nm excitation) was used to determine the overall carotenoid (astaxanthin and cantaxanthin) and fat content in 49 samples of ground muscle tissue from farmed Atlantic salmon (Salmo salar L.). Chemically determined contents ranged from 1.0 to 6.8 mg/kg carotenoids and 36 to 205 g/kg fat. In addition to the raw Raman spectra, three types of spectral preprocessing were evaluated: the first derivative, subtraction of the fitted fourth-order polynomial (POLY), and the intensity normalized versions of POLY (POLY-SNV). Further, variable selection based on significance testing by use of jack-knifing was performed on each spectral data set. Partial least-squares regression resulted in a root mean square error of prediction of 0.33 mg/kg (R = 0.97) for carotenoids for the variable selected versions of all the preprocessed spectral data sets. The fat content was best estimated by the variable selected POLYSNV, resulting in a root mean square error of prediction of 15.5 g/kg (R = 0.95). Both preprocessing and variable selection improved the regression models significantly. The results demonstrate that Raman spectroscopy is a suitable method for simultaneous, rapid, and nondestructive quantification of both pigments and fat in ground salmon muscle tissue.

Topics: Animals; beta Carotene; Canthaxanthin; Chemistry Techniques, Analytical; Lipids; Muscle, Skeletal; Salmo salar; Spectrum Analysis, Raman; Xanthophylls

1. The experiments were conducted to evaluate astaxanthin (Ax) uptake in several tissues and plasma lipoproteins of male broiler chickens fed on Phaffia rhodozyma containing a high concentration of Ax. 2. Male broiler chicks (5 weeks of age) fasted for 16h were given 0 or 45 mg Ax as Phaffia rhodozyma through the crop and blood was collected over the following 24 h. Ax appeared in the plasma at 2 h after administration into the crop. Most (more than 70%) of the Ax was contained in the high density lipoprotein (HDL) fraction in the plasma irrespective of blood sampling times and administration procedure of Ax. 3. Male broiler chicks (2 weeks of age) were fed on a diet containing 0, 50 or 100 mg/kg of yeast Ax for 2 weeks. Of the tissues examined, Ax concentration in the small intestine was highest, followed by subcutaneous fat, abdominal fat, spleen, liver, heart, kidney and skin. The lowest concentration was in the muscles. Ax concentration in the small intestine, subcutaneous fat, abdominal fat, liver and skin rose as dietary content increased, but this was not the case for the spleen, heart, kidney and muscles except for M. pecloralis superficialis. 4. Over 50% of Ax deposited in liver tissues was detected in the microsomal fraction and 15% was in the mitochondrial fraction. In muscles, both fractions of mitochondria and sarcoplasmic reticulum contained Ax.

Topics: Animals; Basidiomycota; beta Carotene; Chickens; Lipoproteins; Liver; Male; Microsomes, Liver; Mitochondria, Liver; Pectoralis Muscles; Tissue Distribution; Xanthophylls

The effects of orally administered carotenoids from natural sources on the non-specific defense mechanisms of rainbow trout were evaluated in a nine-week feeding trial. Fish were fed four diets containing either beta-carotene or astaxanthin at 100 and 200 mg kg-1 from the marine algae Dunaliella salina and red yeast Phaffia rhodozyma, respectively, and a control diet containing no supplemented carotenoids. Specific growth rate and feed:gain ratio were not affected by dietary carotenoid supplementation. Among the humoral factors, serum alternative complement activity increased significantly in all carotenoid supplemented groups when compared to the control. On the other hand, serum lysozyme activity increased in the Dunaliella group but not in the Phaffia group, whereas plasma total immunoglobulin levels were not altered by the feeding treatments. As for the cellular responses, the superoxide anion production from the head kidney remained unchanged while the phagocytic rate and index in all supplemented groups were significantly higher than those of the control. These findings demonstrate that dietary carotenoids from both D. salina and P. rhodozyma can modulate some of the innate defense mechanisms in rainbow trout.

Topics: Analysis of Variance; Animals; Basidiomycota; beta Carotene; Chlorophyta; Chromatography, High Pressure Liquid; Complement Pathway, Alternative; Diet; Immunity, Innate; Immunoglobulins; Muramidase; Oncorhynchus mykiss; Phagocytosis; Superoxides; Xanthophylls

Oral bioavailability of natural and synthetic carotenoids is generally poor in rodents, and this has limited the ability to test these antioxidant compounds in well-defined rodent models of human disease. Various strategies have been employed, with variable success, to increase the percentage of the total oral dose absorbed by the rodent GI tract. In the current study, a novel carotenoid derivative (the disodium disuccinate diester of astaxanthin; Heptax) was administered by oral gavage in a lipophilic emulsion to C57BL/6 mice. Plasma appearance and tissue accumulation of non-esterified, free astaxanthin was studied by HPLC over 72 h after single- and multiple-dose regimens. One-time dosing of Heptax in emulsion at 500 mg/kg resulted in significant appearance of free astaxanthin in plasma (Cmax=0.2 mg/l; 381 nM) and accumulation in solid organs (e.g. liver Cmax=0.9 mg/l; 1735 nM), levels not previously reported after single carotenoid doses in rodents. At each point in the concentration/time curve (AUC), free astaxanthin levels in liver were greater than the corresponding concentration in plasma, suggesting concentrative uptake by the liver. As the ED50 as an antioxidant for non-esterified, free astaxanthin in model systems is approximately 200 nM, the current results suggest that hepatoprotection against oxidative insults may be achieved after a single dose of Heptax in these animals. In humans, where the bioavailability of oral carotenoids ranges from 40 to 60% of the total dose when given in lipophilic vehicle, much smaller oral doses may be utilized for therapeutic benefit in a particular clinical application.

Topics: Administration, Oral; Animals; Area Under Curve; beta Carotene; Chromatography, High Pressure Liquid; Emulsions; Male; Mice; Mice, Inbred C3H; Stereoisomerism; Succinates; Tissue Distribution; Xanthophylls

The expression, at the mRNA level, of carotenoid biosynthetic genes from Xanthophyllomyces dendrorhous was studied by RT-PCR. The experimental conditions for the RT-PCR assay were standardized to quantify the relative transcript levels of idi, crtE, crtYB and crtI genes. This work attempted to correlate astaxanthin production with the transcript levels of carotenogenic genes in a wild-type strain (UCD 67-385) and two overproducer deregulated strains (atxS1 and atxS2). At 3 day cultures, the wild-type strain contained higher transcript levels from the crtE and crtYB genes on minimal medium than on rich medium. Similarly, carotenoid production was higher on minimal medium than on rich medium. However, carotenoid production in the atxS1 and atxS2 strains was not correlated with the transcript level of carotenogenic genes under the same experimental conditions. This result suggests that there is not a linear relationship between carotenogenic transcript levels and carotenoid biosynthesis.

Topics: Basidiomycota; beta Carotene; Culture Media; DNA, Complementary; Gene Expression Regulation, Fungal; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Time Factors; Xanthophylls

Negative ion liquid chromatography-atmospheric pressure chemical ionization mass spectrometry [negative ion LC-(APCI)MS] was used for the identification of astaxanthin esters in extracts of commercial shrimp (Pandalus borealis) and dried microalga (Haematococcus pluvialis) samples. A cleanup step using a normal phase solid phase extraction (SPE) cartridge was applied prior to analysis. Recovery experiments with astaxanthin oleate as model compound proved the applicability of this step (98.5 +/- 7.6%; n = 4). The assignment of astaxanthin esters in negative ion LC-(APCI)MS was based on the detection of the molecular ion (M*-) and the formation of characteristic fragment ions, resulting from the loss of one or two fatty acids. Quantification of individual astaxanthin esters was performed using an astaxanthin calibration curve, which was found to be linear over the required range (1-51 micromol/L; r2 = 0.9996). Detection limits, based on the intensity of M*-, a signal-to-noise ratio of 3:1, and an injection volume of 20 microL, were estimated to be 0.05 microg/mL (free astaxanthin), 0.28 microg/mL (astaxanthin-C16:0), and 0.78 microg/mL (astaxanthin-C16:0/C16:0), respectively. This LC-(APCI)MS method allows for the first time the characterization of native astaxanthin esters in P. borealis and H. pluvialis without using time-consuming isolation steps with subsequent gas chromatographic analyses of fatty acid methyl esters. The results suggest that the pattern of astaxanthin-bound polyunsaturated fatty acids of P. borealis does not reflect the respective fatty acid pattern found in triacylglycerides. Application of the presented LC-(APCI)MS technique in common astaxanthin ester analysis will forestall erroneous xanthophyll ester assignment in natural sources.

Topics: Animals; beta Carotene; Chlorophyta; Chromatography, High Pressure Liquid; Chromatography, Liquid; Esters; Mass Spectrometry; Pandalidae; Xanthophylls

Carotenoids are plant pigments whose consumption is associated with lower cancer rates in humans. Studies in experimental animal and cell systems have confirmed the cancer chemopreventive activity of these compounds. However, their extremely hydrophobic nature makes these compounds biologically unavailable unless delivered in organic solution to model systems. We have synthesized novel disodium salt disuccinate astaxanthin derivatives that possess high aqueous dispersibility. When delivered to mouse embryonic fibroblast C3H/10T1/2 cell cultures, either in aqueous or aqueous/ethanol solutions, these derivatives are biologically active. Biological activity was demonstrated by (1) upregulated expression of connexin 43 (Cx43) protein; (2) increased formation of Cx43 immunoreactive plaques in regions of the plasma membrane consistent with localization of gap junctions; (3) significantly upregulated gap junctional intercellular communication (GJIC) as demonstrated by Lucifer Yellow dye transfer after microinjection (P < 0.03; Fisher's Exact test). Enhanced expression of Cx43 and increased GJIC have been previously demonstrated to result in inhibition of in vitro neoplastic transformation of 10T1/2 cells as well as growth reduction of human tumors in xenografts. These novel derivatives possess increased utility as water soluble and water dispersible agents, allowing for aqueous delivery both in vitro and in vivo, properties that could enhance their potential clinical utility as potent cancer chemopreventive agents.

Topics: Animals; beta Carotene; Cell Communication; Cells, Cultured; Connexin 43; Gap Junctions; Mice; Solutions; Up-Regulation; Xanthophylls

The aqueous solubility and/or dispersibility of synthetic carotenoid analogs can be improved by varying the chemical structure(s) of the esterified moieties. In the current study, a highly water-dispersible astaxanthin (3,3'-dihydroxy-beta,beta-carotene-4,4'-dione) derivative was synthesized by esterification to the amino acid L-lysine, and subsequently converted to the tetrahydrochloride salt. Deep violet, evenly colored aqueous suspensions were obtained with addition of the novel derivative to USP purified water up to a maximum of 181.6 mg/mL. These aqueous suspensions were obtained without the addition of heat, detergents, co-solvents, or other additives. At higher concentrations (above 181.6 mg/mL), the dispersion became turbid and viscous. There was no saturation point up to 181.6 mg/mL. The direct superoxide scavenging ability of the tetrahydrochloride dilysine astaxanthin salt was also evaluated by electron paramagnetic resonance (EPR) spectroscopy in a well-characterized in vitro isolated human neutrophil assay. The novel derivative was an extremely potent (micromolar concentration) aqueous-phase scavenger, with near-complete suppression of the superoxide anion signal (as detected by spin-trap adducts of DEPMPO) achieved at 100 microM. To the authors' knowledge, this novel carotenoid derivative exhibits the greatest aqueous dispersibility yet described for a natural and/or synthetic C40 carotenoid, and as such, will find utility in those applications for which aqueous-phase singlet oxygen quenching and direct radical scavenging are required.

Topics: Amino Acids; beta Carotene; Free Radical Scavengers; Humans; In Vitro Techniques; Models, Molecular; Molecular Conformation; Neutrophils; Solubility; Structure-Activity Relationship; Superoxides; Xanthophylls

Five groups of salmon, of initial mean weight 127 +/- 3 g, were fed increasing levels of dietary linseed oil (LO) in a regression design. The control diet contained capelin oil (FO) only, and the same oil was blended with LO to provide the experimental diets. After an initial period of 40 wk, all groups were switched to a finishing diet containing only FO for a further 24 wk. Growth and flesh lipid contents were not affected by dietary treatment. The FA compositions of flesh total lipids were linearly correlated with dietary FA compositions (r2 = 0.88-1.00, P < 0.0001). LO included at 50% of added dietary lipids reduced flesh DHA and EPA (20:5n-3) concentrations to 65 and 58%, respectively, of the concentrations in fish fed FO. Feeding 100% LO reduced flesh DHA and EPA concentrations to 38 and 30%, respectively, of the values in fish fed FO. Differences between diet and flesh FA concentrations showed that 16:0, 18:1n-9, and especially DHA were preferentially retained in flesh, whereas 18:2n-6, 18:3n-3, and 22:1n-11 were selected against and presumably utilized for energy. In fish previously fed 50 and 100% LO, feeding a finishing diet containing FO for 16 wk restored flesh DHA and EPA concentrations, to approximately 80% of the values in fish fed FO throughout. Flesh DHA and EPA concentrations in fish fed up to 50% LO were above recommended intake values for humans for these EFA. This study suggests that LO can be used as a substitute for FO in seawater salmon feeds and that any reductions in DHA and EPA can be largely overcome with a finishing diet high in FO before harvest.

Topics: Animal Feed; Animals; beta Carotene; Fatty Acids; Fatty Acids, Omega-3; Fish Oils; Linseed Oil; Muscles; Salmo salar; Xanthophylls

To isolate a carotenoid-hyperproducing yeast, Phaffia rhodozyma was treated by low-dose gamma irradiation below 10 kGy. Through repeated rounds of gamma irradiation and visual screening, a mutant 3A4-8 was isolated. It produced 3.3 mg of carotenoid per gram of yeast, 50% higher carotenoid content than that of the unirradiated strain. Glucose and peptone were the most suitable carbon and nitrogen sources for production of carotenoid based on the growth experiment of the mutant under various carbon and nitrogen sources. This result suggests that low-dose gamma irradiation could be used as a means of mutagenesis for isolation of a carotenoid-hyperproducing strain of P. rhodozyma because only the carotenoid-hyperproducing yeast survives gamma irradiation by scavenging oxygen radicals generated by radiolysis of water.

Topics: beta Carotene; Carotenoids; Dose-Response Relationship, Radiation; Gamma Rays; Mutation; Xanthophylls; Yeasts

For the heterologous synthesis of keto-carotenoids such as astaxanthin, two carotenoid ketolase genes crtW38 and crtW148, were cloned from the cyanobacterium, Nostoc punctiforme PCC 73102 and functionally characterized. Upon expression in Escherichia coli, both genes mediated the conversion of beta-carotene to canthaxanthin. However in a zeaxanthin-producing E. coli, only the gene product of crtW148 introduced 4-keto groups into the 3,3'-dihydroxy carotenoid zeaxanthin yielding astaxanthin. The gene product of crtW38 was unable to catalyze this reaction. Both ketolases differ in their interaction with a hydroxylase in the biosynthetic pathway from beta-carotene to astaxanthin.

Topics: Amino Acid Sequence; Bacterial Proteins; beta Carotene; Canthaxanthin; Carotenoids; Cloning, Molecular; Escherichia coli; Gene Expression Regulation, Bacterial; Gene Expression Regulation, Enzymologic; Molecular Sequence Data; Nostoc; Oxygenases; Protein Engineering; Recombinant Proteins; Xanthophylls

Natural carbon sources, such as those present in cane sugar molasses and grape juice, promote the synthesis of astaxanthin in different Phaffia rhodozyma yeasts. One of these, coconut milk, has a very rich nutrient composition. The aim of this work was to investigate the utility of coconut milk as sole source of energy for astaxanthin pigment production by P. rhodozyma strains. Currently, coconut pulp is widely used in industrial processes in Mexico for the production of shampoos, candies, food, etc. However, coconut milk is a waste product. We show that coconut milk enhances astaxanthin production. The fermentation yielded 850 microg/g yeast with the NRRL-10921 wild-type strain and 1850 microg/g yeast with the mutated R1 strain. Production was better than reported results employing other natural carbon sources.

Topics: Basidiomycota; beta Carotene; Cocos; Culture Media; Xanthophylls

An aerobic, non-motile, Gram-negative, orange-pigmented, rod-shaped, astaxanthin-producing marine bacterium was isolated from the Haeundae Coast, Korea. This strain, BC74171T, produced carotenoids, mainly astaxanthin. All the type strains of the genus Paracoccus were compared with strain BC74171T using 16S rRNA gene sequence analysis, fatty acid patterns and physiological reaction profiles. Based on the results of these analyses, it is proposed that strain BC74171T represents a novel species, Paracoccus haeundaensis sp. nov. The type strain is BC74171T (= KCCM 10460T = LMG P-21903T).

Topics: beta Carotene; DNA, Bacterial; DNA, Ribosomal; Fatty Acids; Genes, rRNA; Korea; Microscopy, Electron, Transmission; Molecular Sequence Data; Paracoccus; Phylogeny; Pigments, Biological; RNA, Bacterial; RNA, Ribosomal, 16S; Seawater; Sequence Analysis, DNA; Water Microbiology; Xanthophylls

The composition of atherosclerotic plaques, not just macroscopical lesion size, has been implicated in their susceptibility to rupture and the risk of thrombus formation. By focusing on the quality of lipids, macrophages, apoptosis, collagen, metalloproteinase expression and plaque integrity, we evaluated the possible anti-atherosclerotic effect of the antioxidants alpha-tocopherol and astaxanthin in Watanabe heritable hyperlipidemic (WHHL) rabbits. Thirty-one WHHL rabbits were divided into three groups and were fed a standard diet, as controls (N =10), or a standard diet with the addition of 500 mg alpha-tocopherol per kg feed (N =11) or 100 mg astaxanthin per kg feed (N =10) for 24 weeks. We found that both antioxidants, particularly astaxanthin, significantly decreased macrophage infiltration in the plaques although they did not affect lipid accumulation. All lesions in the astaxanthin-treated rabbits were classified as early plaques according to the distribution of collagen and smooth muscle cells. Both antioxidants also improved plaque stability and significantly diminished apoptosis, which mainly occurred in macrophages, matrix metalloproteinase three expressions and plaque ruptures. Although neither antioxidant altered the positive correlations between the lesion size and lipid accumulation, the lesion size and apoptosis were only positively correlated in the control group. Astaxanthin and alpha-tocopherol may improve plaque stability by decreasing macrophage infiltration and apoptosis in this atherosclerotic setting. Apoptosis reduction by alpha-tocopherol and astaxanthin may be a new anti-atherogenic property of these antioxidants.

Topics: alpha-Tocopherol; Animals; Antioxidants; Aorta, Thoracic; Apoptosis; Arteriosclerosis; beta Carotene; Cell Movement; Collagen; Hyperlipidemias; Lipid Peroxidation; Lipids; Macrophages; Matrix Metalloproteinase 3; Rabbits; Xanthophylls

Appearance, pharmacokinetics and distribution of astaxanthin all-E-, 9Z- and 13Z-geometrical and (3R,3'R)-, (3R,3'S)- and (3S,3'S)-optical isomers in plasma fractions were studied in three middle-aged male volunteers (41-50 years) after ingestion of a single meal containing first a 10-mg dose equivalent of astaxanthin from astaxanthin diesters, followed by a dose of 100 mg astaxanthin equivalents after 4 weeks. Direct resolution of geometrical isomers and optical isomers of astaxanthin dicamphanates by HPLC after saponification showed that the astaxanthin consisted of 95.2% all-E-, 1.2% 9Z- and 3.6% 13Z-astaxanthin, of (3R,3'R)-, (3R,3'S; meso)- and (3S,3'S)-astaxanthin in a 31:49:20 ratio. The plasma astaxanthin concentration-time curves were measured during 76 h. Astaxanthin esters were not detected in plasma. Maximum levels of astaxanthin (C(max)=0.28+/-0.1 mg/l) were reached 11.5 h after administration and the plasma astaxanthin elimination half-life was 52+/-40 h. The C(max) at the low dose was 0.08 mg/l and showed that, the dose response was non-linear. The (3R,3'R)-astaxanthin optical isomer accumulated selectively in plasma compared to the (3R,3'S)- and (3S,3'S)-isomers, and comprised 54% of total astaxanthin in the blood and only 31% of total astaxanthin in the administered dose. The astaxanthin Z-isomers were absorbed selectively into plasma and comprised approximately 32% of total astaxanthin 6-7.5 h postprandially. The proportion of all-E-astaxanthin was significantly higher in the very low density lipoproteins and chylomicrons (VLDL/CM) plasma lipoprotein fraction than in the high density lipoproteins (HDL) and low denisty lipoproteins (LDL) fractions (P<0.05). The results indicate that a selective process increase the relative proportion of astaxanthin Z-isomers compared to the all-E-astaxanthin before uptake in blood and that the astaxanthin esters are hydrolyzed selectively during absorption.

Topics: Administration, Oral; Adult; beta Carotene; Carotenoids; Chromatography, High Pressure Liquid; Esters; Fatty Acids; Humans; Isomerism; Male; Middle Aged; Molecular Structure; Time Factors; Xanthophylls

Epidemiological studies suggest that consumption of vegetables rich in the xanthophylls lutein (LUT) and zeaxanthin (ZEA) reduces the risk for developing age-related cataract, a leading cause of vision loss. Although LUT and ZEA are the only dietary carotenoids present in the lens, direct evidence for their photoprotective effect in this organ is not available. The present study examined the effects of xanthophylls and alpha-tocopherol (alpha-TC) on lipid peroxidation and the mitogen-activated stress signaling pathways in human lens epithelial (HLE) cells following ultraviolet B light (UVB) irradiation. When presented with LUT, ZEA, astaxanthin (AST), and alpha-TC as methyl-beta-cyclodextrin complexes, HLE cells accumulated the lipophiles in a concentration- and time-dependent manner with uptake of LUT exceeding that of ZEA and AST. Pretreatment of cultures with either 2 micromol/L xanthophyll or 10 micromol/L alpha-TC for 4 h before exposure to 300 J/m(2) UVB radiation decreased lipid peroxidation by 47-57% compared with UVB-treated control HLE cells. Pretreatment with the xanthophylls and alpha-TC also inhibited UVB-induced activation of c-JUN NH(2)-terminal kinase (JNK) and p38 by 50-60 and 25-32%, respectively. There was substantial inhibition of UVB-induced JNK and p38 activation for cells containing <0.20 and approximately 0.30 nmol xanthophylls/mg, respectively, whereas >2.3 nmol alpha-TC/mg protein was required to significantly decrease UVB-induced stress signaling. These data suggest that xanthophylls are more potent than alpha-TC for protecting human lens epithelial cells against UVB insult.

Topics: alpha-Tocopherol; beta Carotene; Epithelial Cells; Humans; JNK Mitogen-Activated Protein Kinases; Kinetics; Lens, Crystalline; Lipid Peroxidation; Oxidative Stress; p38 Mitogen-Activated Protein Kinases; Ultraviolet Rays; Xanthophylls

Four groups of rainbow trout (Onchorhynchus mykiss) each with 60 females with an average body weight of 467 g were submitted to an aleatory assay in order to compare the efficiency of four diets containing different concentrations of carotenoid pigments, and to determine if pigment concentration and source had an influence on female gonad development and oocyte maturity. The first diet was the non-pigmented control (C). The second diet contained 100 mg kg(-1) of Carophyll Pink (CR). The third and fourth diets contained 200 (RC200) and 250 (RC250) mg kg(-1) of saponified red chili oleoresin, respectively. The results indicated significant differences (p < or = 0.05) between C and RC250, with respect to the gonado-somatic index (IGS), average gonad weight (PG) and average diameter of oocyte (DO), with CR250 showing higher IGS, PG and DO values. Treatments CR and CR200 presented similar values. At the end of the experimental period, only 10% of oocytes were mature in the RC250 diet.

Topics: Adjuvants, Immunologic; Animals; beta Carotene; Carotenoids; Diet; Female; Gonads; Oncorhynchus mykiss; Oocytes; Plant Extracts; Sexual Maturation; Xanthophylls

The red-colored chlorophyte Chlamydomonas nivalis is commonly found in summer snowfields. We used a modified Li-Cor gas-exchange system to investigate surface gas-exchange characteristics of snow colonized by this alga, finding rates of CO(2) uptake up to 0.3 micromol.m(-2).s(-1) in dense algal blooms. Experiments varying the irradiance resulted in light curves that resembled those of the leaves of higher plants. Red light was more effective than white and much more effective than green or blue, because of the red astaxanthin that surrounds and masks the algal chloroplasts. Integrating daily course measurements of gas exchange showed CO(2) uptake around 2,300 micromol.m(-2).day(-1) in heavily colonized patches, indicating that summer snowfields can be surprisingly productive.

Topics: Animals; beta Carotene; Carbon Dioxide; Chlamydomonas; Chloroplasts; Light; Maryland; Rhodophyta; Xanthophylls

Dietary antioxidants may attenuate oxidative damage from strenuous exercise in various tissues. Beneficial effects of the antioxidant astaxanthin have been demonstrated in vitro, but not yet in vivo. We investigated the effect of dietary supplementation with astaxanthin on oxidative damage induced by strenuous exercise in mouse gastrocnemius and heart. C57BL/6 mice (7 weeks old) were divided into groups: rested control, intense exercise, and exercise with astaxanthin supplementation. After 3 weeks of exercise acclimation, both exercise groups ran on a treadmill at 28 m/min until exhaustion. Exercise-increased 4-hydroxy-2-nonenal-modified protein and 8-hydroxy-2'-deoxyguanosine in gastrocnemius and heart were blunted in the astaxanthin group. Increases in plasma creatine kinase activity, and in myeloperoxidase activity in gastrocnemius and heart, also were lessened by astaxanthin. Astaxanthin showed accumulation in gastrocnemius and heart from the 3 week supplementation. Astaxanthin can attenuate exercise-induced damage in mouse skeletal muscle and heart, including an associated neutrophil infiltration that induces further damage.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Adjuvants, Immunologic; Animals; Antioxidants; beta Carotene; Creatine Kinase; Deoxyguanosine; Dietary Supplements; Female; Immunohistochemistry; Mice; Mice, Inbred C57BL; Muscle, Skeletal; Myocardium; Oxidative Stress; Peroxidase; Physical Conditioning, Animal; Xanthophylls

Carotenoids are the most widely distributed natural pigments, with over 600 individual compounds identified and characterized from natural sources. A few are commercially important molecules, having found utility as additions to animal feed in the aquaculture, poultry, and swine feed industries. The majority are lipophilic molecules with near zero inherent aqueous solubility. Many different methods have been developed to make the carotenoids "water dispersible," as true water solubility has not been described. Astaxanthin (3,3'-dihydroxy-beta, beta-carotene-4,4'-dione) is a commercially important oxygenated carotenoid that has gained wide acceptance as a feed additive in the $50 billion salmon and trout aquaculture industry. Recently, interest in the human health applications of astaxanthin has increased, with astaxanthin receiving approval as a dietary supplement in several countries, including the United States. Moving astaxanthin into a pharmaceutical application will require a chemical delivery system that overcomes the problems with parenteral administration of a highly lipophilic, low molecular weight compound. In the current study, the ability of sulfobutyl ether beta-cyclodextrin (sodium), as the Captisol(R) brand, to increase the aqueous water solubility of crystalline astaxanthin was evaluated. Complexation of crystalline astaxanthin with Captisol increased the apparent water solubility of crystalline astaxanthin approximately 71-fold, to a concentration in the 2 microg/mL range. It is unlikely that this increase in solubility will result in a pharmaceutically acceptable chemical delivery system for humans. However, the increased aqueous solubility of crystalline astaxanthin to the range achieved in the current study will likely find utility in the introduction of crystalline astaxanthin into mammalian cell culture systems that have previously been dependent upon liposomes, or toxic organic solvents, for the introduction of carotenoids into aqueous solution.

Topics: Adjuvants, Immunologic; beta Carotene; beta-Cyclodextrins; Chemistry, Pharmaceutical; Crystallization; Cyclodextrins; Pharmaceutical Solutions; Solubility; Water; Xanthophylls

Fully synchronised germination of Haematococcus pluvialis astaxanthin-replete aplanospores was induced by transfer to nitrogen-sufficient conditions under either high or low light intensities, and growth, pigment content and nitrogen consumption were monitored during the cell cycle. No germination of the aplanospores was achieved in the absence of nitrate, even when cells were transferred at low light intensities. On the other hand, cell density and chlorophyll concentration increased dramatically and astaxanthin concentration decreased in N-sufficient cultures due to the germination of 100% of the aplanospores, as demonstrated by flow cytometry. No significant effect of light intensity was observed on the degradation of astaxanthin during germination. In germinated cultures, nitrogen was depleted more rapidly under high light conditions, which resulted in earlier entry into the aplanospore stage and accumulation of astaxanthin. Germination of aplanospores accompanied by astaxanthin degradation could also be obtained in the dark in nutrient-sufficient conditions although at a much lower efficiency. The results demonstrate that nutrient availability is the main factor controlling the transition between red and green stages of H. pluvialis, with astaxanthin being accumulated only when cell division has ceased. High light levels accelerate the process by increasing the rate of nutrient depletion and providing more energy for astaxanthin synthesis.

Topics: beta Carotene; Chlorophyll; Chlorophyll A; Chlorophyta; Culture Media; Germination; Light; Nitrogen; Spores; Xanthophylls

Astaxanthin (AST) is a carotenoid that is found in marine animals and vegetables. Several previous studies have demonstrated that AST exhibits a wide variety of biological activities including antioxidant, antitumor, and anti-Helicobacter pylori effects. In this study, attention was focused on the antioxidant effect of AST. The object of the present study was to investigate the efficacy of AST in endotoxin-induced uveitis (EIU) in rats. In addition, the effect of AST on endotoxin-induced nitric oxide (NO), prostaglandin E2 (PGE2), and tumor necrosis factor (TNF)-alpha production in a mouse macrophage cell line (RAW 264.7) was studied in vitro.. EIU was induced in male Lewis rats by a footpad injection of lipopolysaccharide (LPS). AST or prednisolone was administered intravenously at 30 minutes before, at the same time as, or at 30 minutes after LPS treatment. The number of infiltrating cells and protein concentration in the aqueous humor collected at 24 hours after LPS treatment was determined. RAW 264.7 cells were pretreated with various concentrations of AST for 24 hours and subsequently stimulated with 10 microg/mL of LPS for 24 hours. The levels of PGE2, TNF-alpha, and NO production were determined in vivo and in vitro.. AST suppressed the development of EIU in a dose-dependent fashion. The anti-inflammatory effect of 100 mg/kg AST was as strong as that of 10 mg/kg prednisolone. AST also decreased production of NO, activity of inducible nitric oxide synthase (NOS), and production of PGE2 and TNF-alpha in RAW264.7 cells in vitro in a dose-dependent manner.. This study suggests that AST has a dose-dependent ocular anti-inflammatory effect, by the suppression of NO, PGE2, and TNF-alpha production, through directly blocking NOS enzyme activity.

Topics: Animals; Antioxidants; Aqueous Humor; beta Carotene; Cell Count; Cell Line; Cell Survival; Dinoprostone; Dose-Response Relationship, Drug; Lipopolysaccharides; Macrophages; Male; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Prednisolone; Rats; Rats, Inbred Lew; Salmonella typhimurium; Tumor Necrosis Factor-alpha; Uveitis, Anterior; Xanthophylls

The carotenoprotein complexes of a freshwater fairy shrimp (Streptocephalus dichotomus) and a daphnid (Moina micrura) were characterised and compared. Based on thin layer chromatography and mass spectral analysis, a variety of cartenoprotein complexes such as astaxanthin, canthaxanthin, antheraxanthin, lutein, beta-cryptoxanthin and violaxanthin were found. Both crustaceans had astaxanthin and canthaxanthin as predominant prosthetic groups. Amino acid analysis of the complexes further revealed high levels of asparagine, glutamine and glycine in both species. Our study highlights the presence of naturally available carotenoid species in both crustaceans and their possible inter-conversion in anostracans.

Topics: Animals; Anostraca; Asparagine; beta Carotene; Canthaxanthin; Carotenoids; Chromatography, Thin Layer; Cladocera; Fresh Water; Glutamine; Glycine; Lutein; Molecular Structure; Proteins; Xanthophylls

The crtYB locus was used as an integrative platform for the construction of specific carotenoid biosynthetic mutants in the astaxanthin-producing yeast Xanthophyllomyces dendrorhous. The crtYB gene of X. dendrorhous, encoding a chimeric carotenoid biosynthetic enzyme, could be inactivated by both single and double crossover events, resulting in non-carotenoid-producing transformants. In addition, the crtYB gene, linked to either its homologous or a glyceraldehyde-3-phosphate dehydrogenase promoter, was overexpressed in the wild type and a beta-carotene-accumulating mutant of X. dendrorhous. In several transformants containing multiple copies of the crtYB gene, the total carotenoid content was higher than in the control strain. This increase was mainly due to an increase of the beta-carotene and echinone content, whereas the total content of astaxanthin was unaffected or even lower. Overexpression of the phytoene synthase-encoding gene (crtI) had a large impact on the ratio between mono- and bicyclic carotenoids. Furthermore, we showed that in metabolic engineered X. dendrorhous strains, the competition between the enzymes phytoene desaturase and lycopene cyclase for lycopene governs the metabolic flux either via beta-carotene to astaxanthin or via 3,4-didehydrolycopene to 3-hydroxy-3'-4'-didehydro-beta-psi-caroten-4-one (HDCO). The monocylic carotenoid torulene and HDCO, normally produced as minority carotenoids, were the main carotenoids produced in these strains.

Topics: Alkyl and Aryl Transferases; Basidiomycota; beta Carotene; Carotenoids; Fungal Proteins; Gene Expression Regulation, Fungal; Genetic Engineering; Geranylgeranyl-Diphosphate Geranylgeranyltransferase; Intramolecular Lyases; Mutation; Oxidoreductases; Recombinant Proteins; Xanthophylls

The unicellular green alga Haematococcus pluvialis accumulates large amounts of the red ketocarotenoid astaxanthin when exposed to various stress situations such as salt stress and high light intensities. Here, the light regulation of Haematococcus carotenoid biosynthesis was examined. Isolation and characterization of the lycopene beta cyclase gene involved in carotenoid biosynthesis was carried out using a functional complementation approach. Subsequently, gene expression of lycopene cyclase, phytoene synthase, phytoene desaturase and carotenoid hydroxylase was analysed in green flagellate cells. All four genes revealed higher transcript levels in response to increased illumination. Not only the induction of astaxanthin biosynthesis but also carotenoid gene expression was found to be correlated with the redox state of the photosynthetic electron transport. In accordance with this result, increased transcript levels for carotenoid biosynthesis genes were detected under both blue and red light conditions. The application of different inhibitors of the photosynthetic electron flow indicated that the photosynthetic plastoquinone pool functions as the redox sensor for the up-regulation of carotenoid biosynthesis genes. These results suggested that in Haematococcus not only the specific astaxanthin pathway but also general carotenoid biosynthesis is subject to photosynthetic redox control.

Topics: Amino Acid Sequence; beta Carotene; Carotenoids; Chlorophyta; DNA, Complementary; Dose-Response Relationship, Radiation; Gene Expression Regulation; Intramolecular Lyases; Light; Molecular Sequence Data; Oxidation-Reduction; Photosynthesis; Sequence Analysis, DNA; Sequence Homology, Amino Acid; Time Factors; Xanthophylls

Xanthophyllomyces dendrorhous (Phaffia rhodozyma) is used as a colorant for aquaculture, egg yolks, and crustaceans but its carotenoids can only be absorbed by animals when its cell wall is degraded. Conditions that degraded the cell wall of X. dendrorhous were developed. To measure the degrees of cell wall degradation, the carotenoid extractability (extracted carotenoid by acetone/total carotenoid) unit was used. Treatment with HCl (0.2 M, 9 h, 90 degrees C) followed by neutralization to pH 3 by NaOH and spray-drying increased carotenoid extractability to 100% with minimal decomposition.

Topics: Basidiomycota; beta Carotene; Biological Availability; Cell Culture Techniques; Cell Extracts; Cell Membrane; Cells, Cultured; Dehydration; Dose-Response Relationship, Drug; Flavoring Agents; Food Additives; Hot Temperature; Hydrochloric Acid; Hydrogen-Ion Concentration; Xanthophylls

Haematococcus pluvialis was mutated by UV or ethyl methanesulphonate. Mutants resistant to nicotine, diphenylamine, fluridone or norflurazon were then selected. Several nicotine-resistant mutants showed increased (1.9% to 2.5% vs. 1.2% w/w) astaxanthin production. Mutants maintained high astaxanthin production over 4 months of repeated culture.

Topics: beta Carotene; Biotechnology; Chlorophyta; Diphenylamine; Ethyl Methanesulfonate; Mutagenesis; Mutation; Nicotine; Pyridazines; Pyridones; Ultraviolet Rays; Xanthophylls

Astaxanthin is a carotenoid with antioxidant properties, synthesised by plants and algae, and distributed in marine seafood. Astaxanthin is also available as a food supplement, but, like other carotenoids, is a very lipophilic compound and has low oral bioavailability. However, bioavailability can be enhanced in the presence of fat. There is not much information in the literature about the pharmacokinetics of oral astaxanthin in humans. In this open parallel study, healthy male volunteers received a single dose of 40 mg astaxanthin, as lipid based formulations or as a commercially available food supplement, followed by blood sampling for further analysis of plasma concentrations. Pharmacokinetic parameters were calculated to evaluate the extent and rate of absorption from each formulation. The elimination half-life was 15.9+/-5.3 h (n=32), and showed a mono-phasic curve. Three lipid based formulations: long-chain triglyceride (palm oil) and polysorbate 80 (formulation A), glycerol mono- and dioleate and polysorbate 80 (formulation B), and glycerol mono- and dioleate, polysorbate 80 and sorbitan monooleate (formulation C), all showed enhanced bioavailability, ranging from 1.7 to 3.7 times that of the reference formulation. The highest bioavailability was observed with formulation B, containing a high content of the hydrophilic synthetic surfactant polysorbate 80.

Topics: Adult; Antioxidants; beta Carotene; Biological Availability; Chemistry, Pharmaceutical; Humans; Kinetics; Male; Metabolic Clearance Rate; Middle Aged; Xanthophylls

Since the yeast Phaffia rhodozyma was first described some 35 years ago, there has been significant interest in the development of commercial processes to exploit its ability to produce carotenoids (approximately 80% astaxanthin). However, the optimal conditions for carotenoid production are not well understood. A key limitation has been the lack of an appropriate sensor for on-line carotenoid quantification. In this study, an in situ Raman spectroscopy probe was used to monitor intracellular carotenoid production for three consecutive P. rhodozyma fed-batch experiments. Raman spectroscopy is particularly well suited to the study of carotenoids due to a resonance effect, which greatly enhances the intensity of the three fundamental carotenoid bands, nu(1) (1513 cm(-1), C(-) (-)C stretch), nu(2) (1154 cm(-1), C-C stretch), and nu(3) (1003 cm(-1), CH(3) rock). For all three cultures, the peak height of these bands was linearly correlated with intracellular carotenoid content (1 to 45 mg/L) to a precision of better than 5%, and the correlation from one experiment was directly applicable to others.

Topics: Algorithms; beta Carotene; Bioreactors; Carotenoids; Computer Simulation; Culture Media; Spectrum Analysis, Raman; Time Factors; Xanthophylls; Yeasts

Carotenoids are a related group of greater than 600 natural compounds, irrespective of geometric- and stereoisomers, with demonstrated antioxidant efficacy. The carotenoids are broadly divided into "carotenes," or non-oxygen substituted hydrocarbon carotenoids, and "xanthophylls," oxygen-substituted carotenoids. The natural compounds are excellent singlet oxygen quenchers as well as lipid peroxidation chain-breakers; this dual antioxidant capacity is generally attributed to the activity of the polyene chain, and increases with the number of conjugated double bonds along the polyene chain length. However, the poor aqueous solubility of most carotenes and the vast majority of xanthophylls limits their use as aqueous-phase singlet oxygen quenchers and direct radical scavengers. A variety of introduction vehicles (e.g., organic solvents, cyclodextrins) have been used to introduce the insoluble carotenoids into aqueous test systems. Hawaii Biotech, Inc. (HBI) successfully synthesized a novel carotenoid derivative, the disodium disuccinate derivative of astaxanthin (3,3(')-dihydroxy-beta,beta-carotene-4,4(')-dione) in all-trans (all-E) form. The novel derivative is a water-dispersible symmetric chiral molecule with two chiral centers, yielding four stereoisomeric forms: 3R,3(')R and 3S,3(')S (enantiomers), and the diastereomeric meso forms (3R,3(')S and 3(')R,3S). The individual stereoisomers were synthesized at high purity (>90% by HPLC) and compared directly for efficacy with the statistical mixture of stereoisomers obtained from the synthesis from the commercial source of astaxanthin (1:2:1 ratio of 3S,3(')S, meso, and 3R,3(')R, respectively). Direct scavenging of superoxide anion was evaluated in a standard in vitro isolated human neutrophil assay by electron paramagnetic resonance (EPR) imaging, employing the spin-trap DEPMPO. Each novel derivative was tested in pure aqueous formulation and in ethanolic formulation shown to completely disaggregate the compounds in solution. In each case, the ethanolic formulation was a more potent scavenging vehicle. No significant differences in scavenging efficiency were noted among the individual stereoisomers and the statistical mixture of stereoisomers, suggesting that the polyene chain alone was responsible for superoxide scavenging. Dose-ranging revealed that the statistical mixture of stereoisomers of the novel derivative, at millimolar (mM) concentrations, could nearly completely eliminate the superoxide anion sig

Topics: beta Carotene; Cyclic N-Oxides; Data Interpretation, Statistical; Electron Spin Resonance Spectroscopy; Free Radical Scavengers; Humans; Neutrophils; Spin Labels; Stereoisomerism; Succinates; Superoxides; Xanthophylls

While humans have taken limited advantage of natural populations of microalgae for centuries (Nostoc in Asia and Spirulina in Africa and North America for sustenance), it is only recently that we have come to realize the potential of microalgal biotechnology. Microalgal biotechnology has the potential to produce a vast array of products including foodstuffs, industrial chemicals, compounds with therapeutic applications and bioremediation solutions from a virtually untapped source. From an industrial (i.e. commercial) perspective, the goal of microalgal biotechnology is to make money by developing marketable products. For such a business to succeed the following steps must be taken: identify a desirable metabolite and a microalga that produces and accumulates the desired metabolite, establish a large-scale production process for the desired metabolite, and market the desired metabolite. So far, the commercial achievements of microalgal biotechnology have been modest. Microalgae that produce dozens of desirable metabolites have been identified. Aided by high throughput screening technology even more leads will become available. However, the successes in large-scale production and product marketing have been few. We will discuss those achievements and difficulties from the industrial point of view by considering examples from industry, specially our own experience at Mera Pharmaceuticals.

Topics: beta Carotene; Biological Factors; Bioreactors; Biotechnology; Commerce; Drug Industry; Eukaryota; Marine Biology; Pharmaceutical Preparations; Xanthophylls

The development of cataracts in Atlantic salmon, Salmo salar L., was studied in 16 groups of smolts fed diets differing in prooxidant (iron, copper, manganese) and antioxidant (vitamin E, vitamin C, astaxanthin) composition and lipid level for 23 weeks in sea water, using a 2(7-3) reduced factorial design. The seven dietary variables were systematically varied at low (requirement level and 150 g lipid kg(-1)) and high levels (below known toxic levels and 320 g lipid kg(-1)). A mean endpoint cataract incidence of approximately 36% was observed. High dietary levels of vitamin C and astaxanthin reduced cataract frequency, whereas high dietary lipid level, iron and manganese were associated with increased cataract frequencies. Considering the nutritional status of selected organs of the fish, only the status of ascorbic acid correlated negatively to cataract development (P < 0.05). The lens glutathione (GSH) status was not correlated to cataract frequency, nor statistically explained by the dietary variables. However, the study shows that balancing the diet with respect to pro- and antioxidant nutrients may significantly protect Atlantic salmon against development of cataracts. An incidence of reversible osmotic cataract observed at week 14 was positively correlated to plasma glucose concentration.

Topics: Animals; Antioxidants; Aqueous Humor; Ascorbic Acid; beta Carotene; Blood Glucose; Cataract; Copper; Diet; Dietary Fats; Eye; Fish Diseases; Glucose; Glutathione; Incidence; Iron; Lipids; Manganese; Nutritional Status; Reactive Oxygen Species; Salmo salar; Vitamin E; Xanthophylls; Zinc

Astaxanthin, a carotenoid without vitamin A activity, has shown anti-oxidant and anti-inflammatory activities; however, its molecular action and mechanism have not been elucidated. We examined in vitro and in vivo regulatory function of astaxanthin on production of nitric oxide (NO) and prostaglandin E2 (PGE2) as well as expression of inducible NO synthase (iNOS), cyclooxygenase-2, tumor necrosis factor-alpha (TNF-alpha), and interleukin-1beta (IL-1beta). Astaxanthin inhibited the expression or formation production of these proinflammatory mediators and cytokines in both lipopolysaccharide (LPS)-stimulated RAW264.7 cells and primary macrophages. Astaxanthin also suppressed the serum levels of NO, PGE2, TNF-alpha, and IL-1beta in LPS-administrated mice, and inhibited NF-kappaB activation as well as iNOS promoter activity in RAW264.7 cells stimulated with LPS. This compound directly inhibited the intracellular accumulation of reactive oxygen species in LPS-stimulated RAW264.7 cells as well as H2O2-induced NF-kappaB activation and iNOS expression. Moreover, astaxanthin blocked nuclear translocation of NF-kappaB p65 subunit and I(kappa)B(alpha) degradation, which correlated with its inhibitory effect on I(kappa)B kinase (IKK) activity. These results suggest that astaxanthin, probably due to its antioxidant activity, inhibits the production of inflammatory mediators by blocking NF-kappaB activation and as a consequent suppression of IKK activity and I(kappa)B-alpha degradation.

Topics: Active Transport, Cell Nucleus; Adjuvants, Immunologic; Animals; beta Carotene; Cell Line; Cyclooxygenase 2; Dinoprostone; Female; Gene Expression Regulation; I-kappa B Kinase; I-kappa B Proteins; Interleukin-1; Isoenzymes; Lipopolysaccharides; Macrophages; Mice; Mice, Inbred BALB C; NF-kappa B; NF-KappaB Inhibitor alpha; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Promoter Regions, Genetic; Prostaglandin-Endoperoxide Synthases; Protein Serine-Threonine Kinases; Reactive Oxygen Species; Tumor Necrosis Factor-alpha; Xanthophylls

Krill is a major source of astaxanthin, which has strong antioxidant activity. Fractions with astaxanthin monoesters and diesters of Antarctic krill Euphausia superba were isolated. Astaxanthin esters were separated by C18-HPLC depending on the number of carbons and double bonds of esterified fatty acid(s). Small amounts of other lipids remained in the samples, but relative molecular masses of carotenoid esters could be measured by field desorption mass spectrometry without fragmentation and interference from contaminant lipids. The fatty acids were determined by calculation of difference between astaxanthin and astaxanthin esters. Only five kinds of fatty acids, dodecanoate, tetradecanoate, hexadecanoate, hexadecenoate and octadecenoate, were detected. Fast atom bombardment mass spectrometry and secondary ion mass spectrometry showed similar spectra. The fatty acid composition in astaxanthin esters was different from those in krill lipids. Therefore, determination of fatty acids in carotenoid esters by a combination of HPLC elution profile and mild mass spectrometry is found to be a useful tool.

Topics: Animals; beta Carotene; Carotenoids; Chromatography, High Pressure Liquid; Esters; Euphausiacea; Fatty Acids; Mass Spectrometry; Xanthophylls

To exploit the promising biochemical activities of naturally-occurring and synthetic hydrophobic carotenoids, it is necessary to improve their aqueous solubility. The disodium disuccinate derivative of synthetic meso-astaxanthin was prepared, and its behavior in pH 7.4 buffer solutions in both the presence and absence of fatty acid-free human serum albumin (HSA) was evaluated. The induced circular dichroism (CD) spectra and red-shifted absorption band of the optically inactive ligand as well as the fluorescence quenching of HSA indicated that at low ligand/protein ratios (less than approximately 1:1 ligand/protein), the meso-carotenoid bound to albumin in monomeric form. Based on the current experimental and available structural data for HSA, the binding site was tentatively localized to the large interdomain cleft of HSA. Around a 1:1 meso-carotenoid/HSA molar ratio, characteristic positive-negative bands appeared in the visible region of the CD spectrum, whose amplitudes increased in parallel with the increasing concentration of the ligand. These oppositely-signed Cotton effects are typical for chiral intermolecular exciton coupling between adjacent polyene chains arranged in right-handed assembly. Surprisingly, the magnitude of these induced CD bands continued to increase at high ligand/protein ratios (up to 13:1 meso-carotenoid/HSA). These results suggest the formation of unique, mixed-type carotenoid-albumin assemblies in which the HSA molecules themselves serve as chiral templates for the generation of supramolecular assemblies.

Topics: beta Carotene; Binding Sites; Circular Dichroism; Humans; Models, Molecular; Protein Binding; Protein Conformation; Protein Structure, Secondary; Serum Albumin; Spectrophotometry; Succinates; Xanthophylls

The microalga Haematococcus pluvialis Flotow is one of the natural sources of astaxanthin, a pigment widely used in salmon feed. This study was made to discover optimal conditions for biomass and astaxanthin production in H. pluvialis from Steptoe, Nevada (USA), cultured in batch mode. Growth was carried out under autotrophic (with NaNO3, NH4Cl and urea) and mixotrophic conditions (with 4, 8, 12 mM sodium acetate) under two photon flux densities (PFD) (35 and 85 mumol m-2 s-1). The carotenogenesis was induced by 1) addition of NaCl (0.2 and 0.8%), 2) N-deprivation and 3) high PFD (150 mumol m-2 s-1). Total carotenoids were estimated by spectrophotometry and total astaxanthin by HPLC. Ammonium chloride was the best N-source for growth (k = 0.7 div day-1, 228-258 mg l-1 and 2.0 x 10(5)-2.5 x 10(5) cells ml-1 at both PFD, respectively). With increasing acetate concentration, a slight increment in growth occurred only at 85 mumol m-2 s-1. Light was the best inductive carotenogenic factor, and the highest carotenoid production (4.9 mg l-1, 25.0 pg cell-1) was obtained in cultures pre-grown in nitrate at low light. The NaCl caused an increase in carotenoid content per cell at increasing salt concentrations, but resulted in a high cell mortality and did not produce any increment in carotenoid content per volume compared to cultures grown at 150 mumol m-2 s-1. The highest carotenoid content per cell (22 pg) and astaxanthin content per dry weight (10.3 mg g-1) (1% w/w) were obtained at 85 mumol m-2 s-1 with 0.8% NaCl.

Topics: beta Carotene; Biomass; Biotechnology; Chlorophyta; Chromatography, High Pressure Liquid; Phototropism; Spectrophotometry; Xanthophylls

We investigated the effects of astaxanthin on the antitumor effector activity of natural killer (NK) cells suppressed by stress in mice in order to define the immunological significance of astaxanthin (ASX) when combined with restraint stress treatment. When the mice were treated with restraint stress alone, the total number of spleen cells, and the level NK cell activity per spleen were reduced to a nadir on day 3. The stress also caused a significant increase in the lipid peroxidation of liver tissue. ASX (100 mg/kg/day, p.o., 4 days) improved the immunological dysfunction induced by restraint stress. On the other hand, metastatic nodules were observed in the livers of syngenic DBA/2 mice on day 12 after inoculation of P815 mastocytoma cells. Hepatic metastasis was promoted further by restraint stress when applied on day 3 before the inoculation of P815. Daily oral administration of ASX (1 mg/kg/day, p.o., 14 days) markedly attenuated the promotion of hepatic metastasis induced by restraint stress. These results suggested that astaxanthin improves antitumor immune responses by inhibiting of lipid peroxidation induced by stress.

Topics: Animals; Antineoplastic Agents, Phytogenic; Antioxidants; beta Carotene; Cells, Cultured; Female; Immunosuppressive Agents; Killer Cells, Natural; Lipid Peroxidation; Liver; Liver Neoplasms; Mast-Cell Sarcoma; Mice; Mice, Inbred C57BL; Mice, Inbred DBA; Neoplasm Metastasis; Neoplasm Transplantation; Restraint, Physical; Stress, Psychological; Thiobarbituric Acid Reactive Substances; Tissue Distribution; Tumor Cells, Cultured; Xanthophylls

UV radiation from sunlight is the most potent environmental risk factor in skin cancer pathogenesis. In the present study the ability of an algal extract to protect against UVA-induced DNA alterations was examined in human skin fibroblasts (1BR-3), human melanocytes (HEMAc) and human intestinal CaCo-2 cells. The protective effects of the proprietary algal extract, which contained a high level of the carotenoid astaxanthin, were compared with synthetic astaxanthin. DNA damage was assessed using the single cell gel electrophoresis or comet assay. In 1BR-3 cells, synthetic astaxanthin prevented UVA-induced DNA damage at all concentrations (10 nM, 100 nM, 10 microM) tested. In addition, the synthetic carotenoid also prevented DNA damage in both the HEMAc and CaCo-2 cells. The algal extract displayed protection against UVA-induced DNA damage when the equivalent of 10 microM astaxanthin was added to all three-cell types, however, at the lower concentrations (10 and 100 nM) no significant protection was evident. There was a 4.6-fold increase in astaxanthin content of CaCo-2 cells exposed to the synthetic compound and a 2.5-fold increase in cells exposed to algal extract. In 1BR-3 cells, exposure to UVA for 2 h resulted in a significant induction of cellular superoxide dismutase (SOD) activity, coupled with a marked decrease in cellular glutathione (GSH) content. However pre-incubation (18 h) with 10 microM of the either the synthetic astaxanthin or the algal extract prevented UVA-induced alterations in SOD activity and GSH content. Similarly, in CaCo-2 cells a significant depletion of GSH was observed following UVA-irradiation which was prevented by simultaneously incubating with 10 microM of either synthetic astaxanthin or the algal extract. SOD activity was unchanged following UVA exposure in the intestinal cell line. This work suggests a role for the algal extract as a potentially beneficial antioxidant.

Topics: Antioxidants; beta Carotene; Colonic Neoplasms; Comet Assay; DNA Damage; Eukaryota; Glutathione; Humans; Intestinal Mucosa; Plant Extracts; Superoxide Dismutase; Tumor Cells, Cultured; Ultraviolet Rays; Xanthophylls

This study was designed to assess the effects of dietary carotenoid supplementation on liver and kidney xenobiotic-metabolizing enzymes in the rainbow trout. Twelve rainbow trout (mean weight 266+/-10 g) were assigned to each of three replicate tanks for each of four dietary treatments; astaxanthin, canthaxanthin, negative control and positive control using beta-naphthoflavone, at a target dietary inclusion of 100 mg kg(-1) for each additive. Fish were fed for 3 weeks at a level of 1.2% body wt. day(-1). Serum carotenoid levels were used as indicators of exposure and were not significantly different (P>0.05) between carotenoid-fed trout. Livers and kidney were frozen separately in liquid N(2) by immersion and microsomal fractions from pooled samples (n=3) assayed for xenobiotic-metabolizing enzyme (cytochrome P450 monoxygenase) activities including ethoxyresorufin O-deethylase; methoxyresorufin O-demethylase; pentoxyresorufin O-dealkylase; benzoxyresorufin O-dearylase; and the conjugating enzymes glucuronosyl transferase; and glutathione-s-transferase. Results revealed that carotenoid treatment did not significantly (P>0.05) induce any enzyme system examined. Results are discussed in the context of metabolism of absorbed carotenoids.

Topics: Adjuvants, Immunologic; Animals; Antioxidants; Aryl Hydrocarbon Hydroxylases; beta Carotene; Canthaxanthin; Carotenoids; Enzyme Induction; Female; Kidney; Liver; Male; Oncorhynchus mykiss; Xanthophylls

To investigate the effects of various carotenoids on the proliferation, cell cycle, apoptosis and expression of bcl-2 gene in breast cancer cell MCF-7.. Time and dose effects of individual carotenoids were detected using the MTT assay. The effects of individual carotenoids on cell cycle and the apoptosis were observed by flow cytometry. The expression of bcl-2 mRNA gene was detected using the RT-PCR method.. All 4 carotenoids tested inhibited the proliferation of MCF-7 cell line, but with different potencies. beta-carotene and lycopene were the most active inhibitors (inhibition rate 88.2% and 87.8%, respectively) followed by zeaxanthin and astaxanthin. All 4 carotenoids did not induce cell apoptosis. Cell cycle progression was blocked at G(2)/M phase with 60 micromol/L lycopene and at G(0)/G(1) phase with 60 micromol/L zeaxanthin dipalmitate. Carotenoids down regulated bcl-2 gene expression.. Carotenoids could inhibit the proliferation of human beast cancer MCF-7 cell line in vitro and the action of carotenoids may be worked through different pathways.

Topics: beta Carotene; Breast Neoplasms; Canthaxanthin; Carotenoids; Cell Cycle; Cell Division; Gene Expression Regulation, Neoplastic; Humans; Lycopene; Proto-Oncogene Proteins c-bcl-2; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tumor Cells, Cultured; Xanthophylls; Zeaxanthins

The composition of the fatty acids of astaxanthin esters and the distribution of astaxanthin optical RS isomers in the esterified and unesterified astaxanthin fractions extracted from the meal of the pelagic red crab langostilla (Pleuroncodes planipes; Decapoda, Anomura) were determined. Astaxanthin diesters comprised approximately 70%, monoesterified astaxanthin approximately 12%, and unesterified astaxanthin approximately 10% of total carotenoids, respectively. Unidentified carotenes and minor yellow xanthophylls represented approximately 8% of the total carotenoids. Three astaxanthin diester fractions (ratio 5:4:1) and one monoester fraction were clearly distinguished by thin-layer chromatography, and fatty acid moieties were determined in all of them. Saturated fatty acids accumulated in astaxanthin diesters, but were reduced in the monoester fraction when compared to langostilla crude oil extract (CE). Astaxanthin diesters, but not monoesters were enriched in C16:0 and C18:1n-9, when compared to the CE. Astaxanthin monoesters were rich in polyunsaturated fatty acids (approximately 70% of total fatty acids), in particular C20:5n-3 and C22:6n-3. Acylation of astaxanthin in langostilla seems to be selective rather than specific. The three diesterified astaxanthin fractions of langostilla had a ratio of approximately 3:1:3 between the (3R,3'R)-, (3R,3'S)-, and (3S,3'S)-astaxanthin isomers, whereas in the monoesterified and unesterified fractions the ratio was approximately 4:1:4. The astaxanthin optical RS isomer composition indicates that langostilla is unable to racemize astaxanthin.

Topics: Animals; beta Carotene; Decapoda; Esters; Fatty Acids; Isomerism; Molecular Structure; Xanthophylls

Oxidative stress induced by hyperglycemia possibly causes the dysfunction of pancreatic beta-cells and various forms of tissue damage in patients with diabetes mellitus. Astaxanthin, a carotenoid of marine microalgae, is reported as a strong anti-oxidant inhibiting lipid peroxidation and scavenging reactive oxygen species. The aim of the present study was to examine whether astaxanthin can elicit beneficial effects on the progressive destruction of pancreatic beta-cells in db/db mice--a well-known obese model of type 2 diabetes. We used diabetic C57BL/KsJ-db/db mice and db/m for the control. Astaxanthin treatment was started at 6 weeks of age and its effects were evaluated at 10, 14, and 18 weeks of age by non-fasting blood glucose levels, intraperitoneal glucose tolerance test including insulin secretion, and beta-cell histology. The non-fasting blood glucose level in db/db mice was significantly higher than that of db/m mice, and the higher level of blood glucose in db/db mice was significantly decreased after treatment with astaxanthin. The ability of islet cells to secrete insulin, as determined by the intraperitoneal glucose tolerance test, was preserved in the astaxanthin-treated group. Histology of the pancreas revealed no significant differences in the beta-cell mass between astaxanthin-treated and -untreated db/db mice. In conclusion, these results indicate that astaxanthin can exert beneficial effects in diabetes, with preservation of beta-cell function. This finding suggests that anti-oxidants may be potentially useful for reducing glucose toxicity.

Topics: Adjuvants, Immunologic; Age Factors; Animals; Antioxidants; beta Carotene; Blood Glucose; Diabetes Mellitus, Type 2; Disease Models, Animal; Glucose; Islets of Langerhans; Mice; Mice, Inbred C57BL; Oxidative Stress; Reactive Oxygen Species; Time Factors; Xanthophylls

Water-dispersible beadlets of carotenoids were used as supplements for human umbilical vein endothelial cells (HUVECs), human peripheral blood mononuclear cells (PBMCs) and human monocytes. Stability, cellular association and cytotoxicity of the carotenoid beadlets were compared with carotenoids delivered with tetrahydrofuran (THF). Incubations with lycopene, beta-carotene, lutein and astaxanthin dissolved in THF resulted in a lower stability in the medium, lower cellular association, and a higher standard deviation. Beadlets provided 60, 4, 6, and 2 times greater accumulation of lycopene, beta-carotene, lutein and astaxanthin, respectively, by PBMCs than THF. The cellular association of carotenoids delivered by THF seems to be more carotenoid-specific than when carotenoids are delivered by beadlets. After 48 h of incubation under cell culture conditions all of the four carotenoids (1 microM) delivered by beadlets to the medium showed a reduction less than 30%. In addition, no cytotoxic effect of the carotenoid beadlets or the vehicle alone was detected in a concentration range of 0.5-5 microM. The results show that beadlets are a non-toxic vehicle for supplementing and stabilizing carotenoids in culture media offering a reasonable compromise in term of cell accumulation efficiency.

Topics: beta Carotene; Carotenoids; Cell Death; Cells, Cultured; Drug Stability; Endothelial Cells; Furans; Humans; Leukocytes, Mononuclear; Lutein; Lycopene; Microspheres; Monocytes; Solubility; Umbilical Veins; Water; Xanthophylls

Previous investigations in the rat have shown that the non-provitamin A carotenoid astaxanthin is metabolized into 3-hydroxy-4-oxo-beta-ionone and 3-hydroxy-4-oxo-7,8-dihydro-beta-ionone and, in addition, is a potent CYP1A gene inducer. Here we investigated the metabolism of this compound as well as its capacity to induce CYP genes in primary cultures of human hepatocytes. Free metabolites of 14C-astaxanthin produced in this cellular model were purified by high pressure liquid chromatography (HPLC) and identified by gas chromatography-mass spectrometry (GC-MS) analyses as 3-hydroxy-4-oxo-beta-ionol and 3-hydroxy-4-oxo-beta-ionone. In addition, deconjugation of polar compounds by glusulase and further analyses with HPLC and GC-MS revealed four radiolabeled metabolites including: 3-hydroxy-4-oxo-beta-ionol, 3-hydroxy-4-oxo-beta-ionone, and their reduced forms, 3-hydroxy-4-oxo-7, 8-dihydro-beta-ionol and 3-hydroxy-4-oxo-7,8-dihydro-beta-ionone. The same four metabolites were identified in human plasma from two volunteers who had orally taken 100 mg astaxanthin 24 h before blood collection. In cultured hepatocytes, astaxanthin was a significant inducer of the major cytochrome P450 enzyme, CYP3A4 as well as of CYP2B6, but not of other CYPs, including those from CYP1A and CYP2C families. The lack of autoinduction of astaxanthin metabolism in human hepatocytes suggests that neither CYP3A4 nor CYP2B6 contribute to the formation of metabolites. We conclude that metabolism of astaxanthin and its CYP-inducing capacity are different in humans and in rats. The novel methodology used in our studies could be extended to evaluating the role of metabolites of more important carotenoids such as beta-carotene in differentiation and carcinogenicity.

Topics: Administration, Oral; Aged; beta Carotene; Chromatography, High Pressure Liquid; Cytochrome P-450 Enzyme System; Enzyme Induction; Female; Gas Chromatography-Mass Spectrometry; Gene Expression; Hepatocytes; Humans; Male; Middle Aged; RNA, Messenger; Xanthophylls

Retinoic acids, vitamin A-related compounds, are known to be inhibitors of telomerase. We found that fucoxanthin from the sea alga Petalonia bingamiae is a potent inhibitor of mammalian replicative DNA polymerases (i.e., pol alpha, delta and epsilon). Since fucoxanthin is a carotenoid (provitamin A-related) compound, we characterized the biochemical modes of vitamin A-related compounds including vitamin A and provitamin A in this report. Subsequently, we found that fucoxanthin, all-trans retinal (RAL, vitamin A aldehyde) and all-trans retinoic acid (RA, vitamin A acid) inhibited the activities of replicative DNA polymerases with IC(50) values of 18-190, 14-17 and 8-30 microM, respectively. On the other hand, all-trans retinol (vitamin A) did not influence any of the DNA polymerase activities. RA inhibited not only the activities of pol alpha, delta and epsilon with IC(50) values of 30, 28 and 8 microM, respectively, but of pol beta with an IC(50) value of 27 microM. The tested vitamin A-related compounds did not influence the activities of DNA polymerases from a higher plant, cauliflower, prokaryotic DNA polymerases, or DNA metabolic enzymes such as human immunodeficiency virus type 1 reverse transcriptase, T7 RNA polymerase and bovine deoxyribonuclease I. RAL and RA should be called selective inhibitors of mammalian DNA polymerases including telomerase, and RAL was a specific inhibitor of mammalian replicative DNA polymerases. As expected from these results in vitro, some of them could prevent the growth of NUGC-3 human gastric cancer cells, and especially RAL was a potent antineoplastic agent with an LD(50) value of 19 microM. The cells were halted at G1 phase in the cell cycle by RAL.

Topics: Antineoplastic Agents; beta Carotene; Cell Division; DNA Polymerase beta; DNA Polymerase I; DNA-Directed DNA Polymerase; Dose-Response Relationship, Drug; Enzyme Inhibitors; Flow Cytometry; Humans; Kinetics; Nucleic Acid Synthesis Inhibitors; Octoxynol; Polyethylene Glycols; Retinaldehyde; Serum Albumin, Bovine; Thymine Nucleotides; Tretinoin; Tumor Cells, Cultured; Vitamin A; Xanthophylls

The carotenoid biosynthetic pathway in algae and plants takes place within plastids. In these organelles, carotenoids occur either in a free form or bound to proteins. Under stress, the unicellular green alga Haematococcus pluvialis accumulates secondary carotenoids, mainly astaxanthin esters, in cytoplasmic lipid vesicles up to 4% of its dry mass. It is therefore one of the favored organisms for the biotechnological production of these antioxidative compounds. We have studied the cellular localization and regulation of the enzyme beta-carotene oxygenase in H. pluvialis that catalyzes the introduction of keto functions at position C-4 of the beta-ionone ring of beta-carotene and zeaxanthin. Using immunogold labeling of ultrathin sections and Western blot analysis of cell fractions, we discovered that under inductive conditions, beta-carotene oxygenase was localized both in the chloroplast and in the cytoplasmic lipid vesicles, which are (according to their lipid composition) derived from cytoplasmic membranes. However, beta-carotene oxygenase activity was confined to the lipid vesicle compartment. Because an early carotenogenic enzyme in the pathway, phytoene desaturase, was found only in the chloroplast (Grünewald, K., Eckert, M., Hirschberg, J., and Hagen, C. (2000) Plant Physiol. 122, 1261-1268), a transport of intermediates from the site of early biosynthetic steps in the chloroplast to the site of oxygenation and accumulation in cytoplasmic lipid vesicles is proposed.

Topics: Antioxidants; beta Carotene; Cell Compartmentation; Chlorophyta; Cytoplasm; Diphenylamine; Industrial Microbiology; Oxygenases; Plastids; Pyridazines; Xanthophylls

Astaxanthin is a high-value carotenoid used as a pigmentation source in fish aquaculture. In addition, a beneficial role of astaxanthin as a food supplement for humans is becoming evident. The unicellular green alga Haematococcus pluvialis seems to be a suitable source for natural astaxanthin. Astaxanthin accumulation in H. pluvialis occurs in response to environmental stress such as high light and salt stress. Here, the isolation of the H. pluvialis carotenoid biosynthesis gene phytoene synthase is reported. Furthermore, the expression of phytoene synthase and carotenoid hydroxylase, two key enzymes in astaxanthin biosynthesis, was investigated at the transcriptional level. The application of environmental stress resulted in increased steady-state mRNA levels of both genes. High-light intensity led to a transient increase in carotenoid hydroxylase mRNA followed by moderate astaxanthin accumulation. In contrast, salt stress in combination with high light resulted in a sustained increase in both transcripts. The addition of compounds inducing reactive oxygen species did not influence transcript levels of phytoene synthase and carotenoid hydroxylase. The application of an inhibitor of photosynthesis, 3-(3, 4-dichlorophenyl)-1,1-dimethylurea, indicated that the light-induced expression of these carotenoid biosynthesis genes may be under photosynthetic control.

Topics: Alkyl and Aryl Transferases; beta Carotene; Carotenoids; Chlorophyta; DNA, Complementary; Geranylgeranyl-Diphosphate Geranylgeranyltransferase; Mixed Function Oxygenases; Plasmids; Xanthophylls

The purpose of this study was to investigate if carotenoids could alleviate the adverse effects caused by aflatoxin with respect to growth performance and immune response. In two experiments, a total of 320 mule ducklings were assigned to 5 treatments, i.e. control, aflatoxin B(1) (AFB(1)) 200 ppb, AFB(1) +beta-carotene (BC) 200 ppm, AFB(1)+BC 400 ppm, and AFB(1)+astaxanthin (AS) 200 ppm. In experiment 1, the addition of beta-carotene or astaxanthin in the diet containing AFB(1) 200 ppb resulted in a significant decrease in average daily gain as compared with the control. AFB(1) 200 ppb alone and the addition of BC or AS on top of AFB(1) resulted in a significantly lower daily feed intake than for the control group. There were no significant differences in relative organ weights among treatment groups. Both treatments of BC 400 ppm and AS 200 ppm had significantly more macrophages harvested per duck than the control and AFB(1) 200 ppb treatments. However, there were no significant differences among treatments in percentages of phagocytotic macrophages and number of Candida albican phagocytized by phagocytotic macrophages. In experiment 2, blood biochemical parameters and antibody titers were evaluated. There were no significant differences among treatments in total bilirubin content and alkaline phosphatase activity in the serum or in antibody titers against fowl cholera. However, AFB(1) treatment had the highest activities of AST and ALT in the serum. The addition of BC 400 ppm on top of AFB(1) significantly reduced ALT activity as compared with the AFB(1) 200 ppb treatment. These results suggest that carotenoids could provide a slightly toxic alleviating effect on growth performance, enhance the chemotaxis ability of macrophages, and reduce ALT activity elevated by AFB(1).

Topics: Aflatoxin B1; Alanine Transaminase; Alkaline Phosphatase; Animal Feed; Animals; Antibodies, Bacterial; Antibody Formation; Aspartate Aminotransferases; beta Carotene; Bilirubin; Biomarkers; Candida albicans; Cholera Toxin; Ducks; Energy Intake; Macrophages; Organ Size; Phagocytosis; Reference Values; Weight Gain; Xanthophylls

The influence of ammonium, phosphate and citrate on astaxanthin production by the yeast Phaffia rhodozyma was investigated. The astaxanthin content in cells and the final astaxanthin concentration increased upon reduction of ammonium from 61 mM to 12.9 mM (from 140 microg/g to 230 microg/g and 1.2 microg/ml to 2.3 microg/ml, respectively). Similarly, both the astaxanthin content and astaxanthin concentration increased by reducing phosphate from 4.8 mM to 0.65 mM (160 microg/g to 215 microg/g and 1.7 microg/ml to 2.4 microg/ml, respectively). Low concentrations of ammonium or phosphate also increased the fatty acid content in cells. By analogy with lipid synthesis in other oleaginous yeasts, an examination of the data for varying nitrogen and phosphate levels suggested that citrate could be the source of carbon for fatty acids and carotenoid synthesis. Supporting this possibility was the fact that supplementation of citrate in the medium at levels of 28 mM or higher notably increased the final pigment concentration and pigment content in cells. Increased carotenoid synthesis at low ammonium or phosphate levels, and stimulation by citrate were both paralleled by decreased protein synthesis. This suggested that restriction of protein synthesis could play an important role in carotenoid synthesis by P. rhodozyma.

Topics: beta Carotene; Citric Acid; Culture Media; Phosphates; Quaternary Ammonium Compounds; Xanthophylls; Yeasts

Molecular mechanisms underlying the peculiar spectral properties of the carotenoid astaxanthin in alpha-crustacyanin, the blue carotenoprotein isolated from the exoskeleton of the lobster Homarus gammarus, were investigated by comparing the basic electrooptical parameters of astaxanthin free in vitro with those of astaxanthin in the complex. Absorption and electroabsorption (Stark effect) spectra were obtained for alpha-crustacyanin in low-temperature glasses to provide information about the molecular interactions that lead to the large bathochromic shift of the spectra resulting from this complexation. The low-temperature spectra reveal the presence of at least three spectral forms of alpha-crustacyanin, with vibronic (0-0) transitions at 14000 cm(-1), 13500 cm(-1) and 11600 cm(-1) (corresponding to approximately 630, 660 and 780 nm, respectively, at room temperature) and with relative aboundance 85%, 10% and 5%. The longer wavelength absorbing species have not previously been detected. The changes in polarizability and in permanent dipole moments associated with the S0-->S2 electronic transition for all these forms are about 1.5 times larger than for isolated astaxanthin. The results are discussed with reference to the symmetric polarization model for astaxanthin in alpha-crustacyanin.

Topics: Animals; beta Carotene; Carrier Proteins; Ethylene Glycol; Glycerol; Nephropidae; Proteins; Spectrum Analysis; Xanthophylls

The effects of the carotenoids beta-carotene and astaxanthin on the peroxidation of liposomes induced by ADP and Fe(2+) were examined. Both compounds inhibited production of lipid peroxides, astaxanthin being about 2-fold more effective than beta-carotene. The difference in the modes of destruction of the conjugated polyene chain between beta-carotene and astaxanthin suggested that the conjugated polyene moiety and terminal ring moieties of the more potent astaxanthin trapped radicals in the membrane and both at the membrane surface and in the membrane, respectively, whereas only the conjugated polyene chain of beta-carotene was responsible for radical trapping near the membrane surface and in the interior of the membrane. The efficient antioxidant activity of astaxanthin is suggested to be due to the unique structure of the terminal ring moiety.

Topics: Adenosine Diphosphate; Antioxidants; beta Carotene; Cardiolipins; Ferrous Compounds; Free Radicals; Hydrogen Bonding; Iron; Kinetics; Lipid Peroxidation; Liposomes; Models, Molecular; Molecular Conformation; Oxidation-Reduction; Phosphatidylcholines; Surface Properties; Xanthophylls; Zeaxanthins

The production of ketocarotenoids (KCs) from Chlorococcum sp. strain MA-1 was investigated by a two-step process. In the first step, 18 g biomass l(-1) was achieved by feeding glucose to the heterotrophic cultures; in the second step, the high-density cultures were treated with light illumination or chemical stress in dark, respectively, to induce KC synthesis. Light-treated cultures could produce 103 mg total KCs l(-1) and 32 mg astaxanthin l(-1), three times higher than those from chemical-treated cultures, in the 10 days of induction. The percentages of individual KCs (hydroxyechinenone, canthaxanthin, adonirubin and astaxanthin) in the total KCs were not markedly influenced by the different stress conditions. The developed two-step process provides a feasible strategy for commercial production of ketocarotenoids by the green microalga, Chlorococcum sp. strain MA-1.

Topics: beta Carotene; Biomass; Biotechnology; Canthaxanthin; Carotenoids; Chlorophyta; Glucose; Light; Xanthophylls

The alga Chlamydomonas nivalis lives in a high-light, cold environment: persistent alpine snowfields. Since the algae in snow receive light from all angles, the photon fluence rate is the critical parameter for photosynthesis, but it is rarely measured. We measured photon irradiance and photon fluence rate in the snow that contained blooms of C. nivalis. On a cloudless day the photon fluence rate at the snow surface was nearly twice the photon irradiance, and it can be many times greater than the photon irradiance when the solar angle is low or the light is diffuse. Beneath the surface the photon fluence rate can be five times the photon irradiance. Photon irradiance and photon fluence rate declined exponentially with depth, approximating the Bouguer-Lambert relationship. We used an integrating sphere to measure the spectral characteristics of a monolayer of cells and microscopic techniques to examine the spectral characteristics of individual cells. Astaxanthin blocked blue light and unknown absorbers blocked UV radiation; the penetration of these wavelengths through whole cells was negligible. We extracted astaxanthin, measured absorbance on a per-cell basis and estimated that the layer of astaxanthin within cells would allow only a small percentage of the blue light to reach the chloroplast, potentially protecting the chloroplast from excessive light.

Topics: Animals; beta Carotene; Chlamydomonas; Chloroplasts; Light; Optics and Photonics; Photobiology; Photons; Photosynthesis; Snow; Spectrophotometry; Xanthophylls

Sequential methodology based on the application of three types of experimental designs was used to optimize the astaxanthin production of the mutant strain 25-2 of Phaffia rhodozyma in shake flask cultures. The first design employed was a factorial design 2(5), where the factors studied were: pH, temperature, percent of inoculum, carbon and nitrogen concentrations, each one at two levels. This design was performed in two medium types: rich YM medium and minimal medium, based on date juice (Yucca medium). With this first design the most important factors were determined (carbon concentration and temperature) that were used in the second experimental strategy: the method of steepest ascent was applied in order to rapidly approach the optimum. Finally, a second-order response surface design was applied using temperature and carbon concentration as factors. The optimal conditions stimulating the highest astaxanthin production were: 19.7 degrees C temperature; 11.25 g l(-1) carbon concentration; 6.0 pH; 5% inoculum and 0.5 g l(-1) nitrogen concentration. Under these conditions the astaxanthin production was 8100 microg l(-1), 92% higher than the production under the initial conditions.

Topics: Basidiomycota; beta Carotene; Biotechnology; Fermentation; Models, Biological; Xanthophylls

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 Fe(2+) 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 beta-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 beta-carotene (norflurazon) and fatty acid (cerulenin) synthesis, and of cytochrome P450 enzyme activity (ellipticine).

Topics: beta Carotene; Cerulenin; Chlorophyta; Cytochrome P-450 Enzyme System; Ellipticines; Enzyme Inhibitors; Fatty Acids; Light; Mixed Function Oxygenases; Pyridazines; Xanthophylls; Zeaxanthins

A two-stage culture system was established for the production of astaxanthin from Haematococcus pluvialis. In a first stage green vegetative cells were produced in semicontinuous cultures maintained with daily renewal rates between 10 and 40%. The steady-state cell density decreased with increasing renewal rates. Highest cell productivity, 64 x 10(6) cells l(-1) day(-1) was obtained with a daily renewal rate of 20%. In a second stage the harvested cultures were submitted to high light (240 micromol photon m(-2) s(-1)) under batch conditions for 15 days in order to stimulate the transition to the aplanospore stage and the accumulation of astaxanthin. No decrease in cell density was recorded during the induction period in any of the cultures. Cultures obtained at high renewal rates continued growing during the induction period and no astaxanthin was accumulated until all nitrogen in the media had been consumed. The final concentration of astaxanthin was inversely correlated to the growth rate at which first-stage cultures were maintained. Optimal renewal rate for maximal astaxanthin production depended on the duration of the induction period. After a 12-day induction period the highest astaxanthin production, 5.8 mg l(-1) of semi-continuous culture day -1, was obtained with cultures maintained at a renewal rate of 20%. When the induction period was increased to 15 days maximal astaxanthin productivity, 9.6 mg l(-1) of semi-continuous culture day -1, was obtained from cultures maintained at a renewal rate of 40% despite the much lower astaxanthin concentration achieved in these cultures. Results demonstrate the feasibility of semi-continuous cultivation of H. pluvialis for the two-stage production of astaxanthin.

Topics: beta Carotene; Chlorophyta; Xanthophylls

Astaxanthin is one of many carotenoids present in marine animals, vegetables and fruits. Since carotenoids are known to have antioxidant properties, we tested to determine if astaxanthin could have protective effects in the CCl4-treated rat liver by activating the antioxidant system. Astaxanthin blocked the increase of glutamate-oxalacetate transaminase (GOT) and glutamate-pyruvate transaminase (GTP) activity and thiobarbituric acid reactive substances (TBARS) in response to carbon tetrachloride (CCl4), while causing an increase in glutathione (GSH) levels and superoxide dismutase (SOD) activities in the CCl4-treated rat liver. These results suggest that astaxanthin protects liver damage induced by CCl4 by inhibiting lipid peroxidation and stimulating the cellular antioxidant system.

Topics: Adjuvants, Immunologic; Animals; beta Carotene; Body Weight; Carbon Tetrachloride Poisoning; Chemical and Drug Induced Liver Injury; Glutathione Reductase; Lipid Peroxidation; Liver; Male; Rats; Rats, Sprague-Dawley; Superoxide Dismutase; Thiobarbituric Acid Reactive Substances; Xanthophylls

High-speed counter-current chromatography was applied to the isolation and purification of astaxanthin from microalgae. The crude astaxanthin was obtained by extraction with organic solvents after the astaxanthin esters were saponified. Preparative high-speed counter-current chromatography with a two-phase solvent system composed of n-hexane-ethyl acetate-ethanol-water (5:5:6.5:3, v/v) was successfully performed yielding astaxanthin at 97% purity from 250 mg of the crude extract in a one-step separation.

Topics: beta Carotene; Chlorophyta; Chromatography, High Pressure Liquid; Countercurrent Distribution; Xanthophylls

Astaxanthin and peridinin, two typical carotenoids of marine microalgae, and lycopene were incorporated in phosphatidylcholine multilamellar liposomes and tested as inhibitors of lipid oxidation. Contrarily to peridinin results, astaxanthin strongly reduced lipid damage when the lipoperoxidation promoters-H(2)O(2), tert-butyl hydroperoxide (t-ButOOH) or ascorbate-and Fe(2+):EDTA were added simultaneously to the liposomes. In order to check if the antioxidant activity of carotenoids was also related to their effect on membrane permeability, the peroxidation processes were initiated by adding the promoters to Fe(2+)-loaded liposomes (encapsulated in the inner aqueous solution). Despite that the rigidifying effect of carotenoids in membranes was not directly measured here, peridinin probably has decreased membrane permeability to initiators (t-ButOOH > ascorbate > H(2)O(2)) since its incorporation limited oxidative damage on iron-liposomes. On the other hand, the antioxidant activity of astaxanthin in iron-containing vesicles might be derived from its known rigidifying effect and the inherent scavenging ability.

Topics: Antioxidants; Ascorbic Acid; beta Carotene; Carotenoids; Cell Membrane Permeability; Chromans; Free Radical Scavengers; Hydrogen Peroxide; Iron; Lipid Peroxidation; Liposomes; Models, Biological; Oxidative Stress; Reactive Oxygen Species; tert-Butylhydroperoxide; Thiobarbituric Acid Reactive Substances; Xanthophylls

The molecular characteristics of the monolayers of astaxanthin with polar group on the beta-ionone ring in the molecule and beta-carotene without polar group and their interactions in mixed carotenoid-phospholipid monolayers and the effects of carotenoids on the phase behavior of the phospholipid bilayers were examined by the monolayer technique and differential scanning calorimetry (DSC). We found from the monolayer study that beta-carotene had an amphiphilic nature. The molecular assembly of astaxanthin in the monolayer at the hydrophobic/hydrophilic interface was more stable than that of beta-carotene. Dimyristoylphosphatidylcholine (DMPC) in the monolayer was miscible with astaxanthin in the range of 0-0.4 mol fractions of astaxanthin, but not fully miscible with beta-carotene even at low concentrations below 0.1 mol fraction of beta-carotene. Surface potential and compression/expansion cycles of beta-carotene monolayer indicated the formation of molecular aggregates by itself. DSC study showed that when small amount of astaxanthin was added, the transition temperature of dipalmitoylphosphatidylcholine (DPPC) was markedly shifted to lower temperatures and that the transition peak was asymmetrically broadened, indicative of a significant depression in cooperativity of the gel to liquid-crystalline transition. The asymmetric DSC endothermic bands of DPPC incorporating small amounts of astaxanthin were well fit by deconvolution into two to three domains containing different concentrations of astaxanthin. On the contrary, the incorporation of beta-carotene resulted in a small depression of the main transition temperature with a slight broadening of the transition peak, suggesting a small miscibility of beta-carotene with the phospholipid bilayer or a formation of aggregates of beta-carotene in the membranes. These results suggest that there would be a high localized concentration in the phase separated membrane for astaxanthin or beta-carotene to function effectively as scavenger.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Animals; beta Carotene; Calorimetry, Differential Scanning; Dimyristoylphosphatidylcholine; Dose-Response Relationship, Drug; Humans; Lipid Bilayers; Membrane Microdomains; Micelles; Phospholipids; Temperature; Thermodynamics; Xanthophylls

The study comprised two parts. Firstly, the effects of dietary supplementation with an algal meal (Novasta) with a high astaxanthin content on ovulation rate (number of corpora lutea, implantation rate, number, mass and length of fetuses) of breeding female mink were evaluated. Secondly, reproductive outcome (number of live and stillborn kits), kit growth rate and milk intake were studied. Both studies were performed on standard brown female mink (n = 20; control (n = 10) and experimental (n = 10)) housed under conventional farm conditions. Experimental animals were supplied with 5.35 mg astaxanthin per day (0.25 g algal meal (Novasta)). The numbers of corpora lutea, implantation sites and fetuses appeared to be higher in the group that was given astaxanthin but the effect was not significant. The differences between treated and control mink were 1.4 (corpora lutea), 0.9 (implantation sites) and 1.2 (litter size). The percentage of stillborn kits was reduced by 6.3 (P < 0.005). The milk intake as measured by use of the isotopic water dilution technique was not affected by treatment group. Milk intake increased from about 19 g in week 1 of lactation to about 30 g per kit per day in week 4 of lactation. Kit weight gain was not affected by the experimental treatment.

Topics: Animals; Animals, Newborn; Antioxidants; beta Carotene; Dietary Supplements; Eating; Female; Fetal Death; Litter Size; Milk; Mink; Ovulation; Pregnancy; Pregnancy Outcome; Reproduction; Xanthophylls

The ability of astaxanthin and canthaxanthin as chain-breaking antioxidants was studied in Cu(2+)-initiated peroxidation of phosphatidylcholine large unilamellar vesicles (LUVs). Both carotenoids increased the lag period that precedes the maximum rate of lipid peroxidation, though astaxanthin showed stronger activity. For these experiments, different amounts of xanthophylls were exogenously added to previously made LUVs, non-incorporated pigment being afterwards removed. Differential scanning calorimetry assays with L-beta,gamma-dimyristoyl-alpha-phosphatidylcholine LUVs demonstrated that xanthophylls incorporated as described interact with the lipid matrix becoming interspersed among the phospholipid molecules.

Topics: Antioxidants; beta Carotene; Calorimetry, Differential Scanning; Canthaxanthin; Carotenoids; Dimyristoylphosphatidylcholine; In Vitro Techniques; Lipid Peroxidation; Liposomes; Xanthophylls

The kinetics of reaction between trifluoroacetic acid as an acid of medium strength and the carotenoids beta-carotene, zeaxanthin, canthaxanthin, and astaxanthin has been examined in detail including the effects of dioxygen, acid concentration, and carotenoid structure. Reaction between acid and carotenoid leads to species absorbing in the red and near-infrared (NIR) spectral regions, intermediates that subsequently disappear. ESR experiments clearly show that these species are not carotenoid radicals, although their NIR absorption is similar to the absorption of carotenoid radical cations. Under most reaction conditions, the disappearance of carotenoids follows pseudo-zero-order kinetics, whereas the reaction order is >1 with respect to acid, and the long-lived (hours) intermediates are suggested to be mono- (700 nm) and diprotonated carotenoid ( approximately 950 nm). Acid induces cis/trans-isomerization via the protonated intermediates, which also decay to nonradical species with shorter conjugated systems-most probably carotenoid esters. Slow protonization of the methine carbon is the primary step in the degradation, but dioxygen increases the rate as a result of formation of a charge-transfer complex with the carotenoids as indicated by a red-shift of the NIR absorption bands. Carotenoids with carbonyl groups (astaxanthin and canthaxanthin) have slower rates of degradation than beta-carotene and zeaxanthin, indicating preferential nondegradative protonation of the carbonyl groups.

Topics: beta Carotene; Canthaxanthin; Carotenoids; Electron Spin Resonance Spectroscopy; Hydrogen-Ion Concentration; Kinetics; Oxygen; Trifluoroacetic Acid; Xanthophylls; Zeaxanthins

The effects of carotenoids--alpha-carotene, beta-carotene, lycopene, beta-cryptoxanthin, zeaxanthin, lutein, canthaxanthin, astaxanthin--on the invasion of rat ascites hepatoma AH109A cells were investigated by co-culturing the hepatoma cells with rat mesentery-derived mesothelial cells (M-cells). All the carotenoids examined inhibited AH109A invasion in a dose-dependent manner up to 5 microM. Cancer cells previously cultured with hypoxanthine (HX) and xanthine oxidase (XO) showed a highly invasive activity. Carotenoids, 5 microM of beta-carotene and astaxanthin, suppressed this reactive oxygen species-potentiated invasive capacity by simultaneously treating AH109A cells with the carotenoids, HX and XO. These results suggest that the antioxidative property of these carotenoids may be involved in their anti-invasive action.

Topics: Animals; beta Carotene; Canthaxanthin; Carotenoids; Coculture Techniques; Cryptoxanthins; Epithelial Cells; Liver Neoplasms, Experimental; Lutein; Lycopene; Neoplasm Invasiveness; Rats; Xanthophylls; Zeaxanthins

The antioxidant activities of astaxanthin and related carotenoids have been measured by employing a newly developed fluorometric assay. This assay is based on 4,4-difluoro-3,5-bis(4-phenyl-1, 3-butadienyl)-4-bora-3a,4a-diaza-s-indacene (BODIPY 665/676) as an indicator; 2,2'-azobis-2,4-dimethylvaleronitrile (AMVN) as a peroxyl radical generator; and 6-hydroxy-2,5,7, 8-tetramethylchroman-2-carboxylic acid (Trolox) as a calibrator in an organic and liposomal media. By employing this assay, three categories of carotenoids were examined: namely, the hydrocarbon carotenoids lycopene, alpha-carotene, and beta-carotene; the hydroxy carotenoid lutein; and the alpha-hydroxy-ketocarotenoid astaxanthin. The relative peroxyl radical scavenging activities of Trolox, astaxanthin, alpha-tocopherol, lycopene, beta-carotene, lutein, and alpha-carotene in octane/butyronitrile (9:1, v/v) were determined to be 1.0, 1.0, 1.3, 0.5, 0.4, 0.3, and 0.2, respectively. In dioleoylphosphatidyl choline (DOPC) liposomal suspension in Tri-HCl buffer (pH 7.4 at 40 degrees C), the relative reactivities of astaxanthin, beta-carotene, alpha-tocopherol, and lutein were found to be 1.00, 0.9, 0.6, and 0.6, respectively. When BODIPY 665/676 was replaced by 4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a, 4a-diaza-s-indacene-3-undecanoic acid (BODIPY 581/591 C(11)) as an indicator, astaxanthin showed the highest antioxidant activity toward peroxyl radicals. The relative reactivities of Trolox, astaxanthin, alpha-tocopherol, alpha-carotene, lutein, beta-carotene, and lycopene were determined to be 1.0, 1.3, 0.9, 0.5, 0.4, 0.2, and 0.4, respectively.

Topics: Antioxidants; beta Carotene; Boron Compounds; Calibration; Carotenoids; Fluorescent Dyes; Free Radical Scavengers; Free Radicals; Indicators and Reagents; Peroxides; Spectrometry, Fluorescence; Vitamin E; Xanthophylls

Astaxanthin, a carotenoid without vitamin A activity, may exert antitumor activity through the enhancement of immune responses. Here, we determined the effects of dietary astaxanthin on tumor growth and tumor immunity against transplantable methylcholanthrene-induced fibrosarcoma (Meth-A tumor) cells. These tumor cells express a tumor antigen that induces T cell-mediated immune responses in syngenic mice. BALB/c mice were fed astaxanthin (0.02%, 40 micrograms/kg body wt/day in a beadlet form) mixed in a chemically defined diet starting zero, one, and three weeks before subcutaneous inoculation with tumor cells (3 x 10(5) cells, 2 times the minimal tumorigenic dose). Three weeks after inoculation, tumor size and weight were determined. We also determined cytotoxic T lymphocyte (CTL) activity and interferon-gamma (IFN-gamma) production by tumor-draining lymph node (TDLN) and spleen cells by restimulating cells with Meth-A tumor cells in culture. The astaxanthin-fed mice had significantly lower tumor size and weight than controls when supplementation was started one and three weeks before tumor inoculation. This antitumor activity was paralleled with higher CTL activity and IFN-gamma production by TDLN and spleen cells in the astaxanthin-fed mice. CTL activity by TDLN cells was highest in mice fed astaxanthin for three weeks before inoculation. When the astaxanthin-supplemented diet was started at the same time as tumor inoculation, none of these parameters were altered by dietary astaxanthin, except IFN-gamma production by spleen cells. Total serum astaxanthin concentrations were approximately 1.2 mumol/l when mice were fed astaxanthin (0.02%) for four weeks and appeared to increase in correlation with the length of astaxanthin supplementation. Our results indicate that dietary astaxanthin suppressed Meth-A tumor cell growth and stimulated immunity against Meth-A tumor antigen.

Topics: Adjuvants, Immunologic; Animals; Antigens, Neoplasm; beta Carotene; Diet; Female; Fibrosarcoma; Immunity, Cellular; Interferon-gamma; Lymph Nodes; Mammary Neoplasms, Experimental; Methylcholanthrene; Mice; Mice, Inbred BALB C; Neoplasm Transplantation; Neoplasms, Experimental; Spleen; T-Lymphocytes, Cytotoxic; Xanthophylls

Carotenoid compositions of the flesh, skin, and ovaries were determined in sexually maturing and immature Arctic charr (Salvelinus alpinus) fed diets supplemented with astaxanthin (optical isomer ratio (3S,3'S):(3R,3'S; meso):(3R,3'R); 1:2:1). Astaxanthin comprised 64-79% of the flesh carotenoids, and the 3',4'-cis and 3',4'-trans glycolic isomers of idoxanthin, present in a 1:1 ratio, represented 20-35%. The flesh of the sexually maturing charr contained relatively more idoxanthin than that of sexually immature fish (20 vs 35% of total carotenoids), possibly being indicative of a higher metabolic turnover of astaxanthin in the latter. The relative proportions of flesh carotenoids were unaffected by sex. The relative carotenoid composition of ovaries was similar in sexually maturing and immature females. The 3',4'-cis and 3',4'-trans glycolic isomers of idoxanthin (ratio 0.7:1) were the major carotenoids (56% of total), followed by crustaxanthin (20%), and astaxanthin comprised less than 5% of ovarian carotenoids. Three glycolic isomers of crustaxanthin were detected (3,4,3',4'-di-cis-:3,4-cis-3',4'-trans-:3,4,3',4'-di-trans-glycolic isomer ratio 2.6:3.1:1) in the ovaries. Sex and maturity status had no apparent effect on the relative composition of skin carotenoids. The skin carotenoids consisted mainly of diesters (82-87% of total carotenoids) and monoesters (7-13% of total carotenoids). Saponification revealed that astaxanthin comprised 85% and idoxanthin 10% of total carotenoids, and minor amounts of tunaxanthin-, lutein-, and zeaxanthin-like metabolites were also present. Maturity status seems to be more important than sex in determining the relative carotenoid composition of the tissues of Arctic charr, with astaxanthin and its metabolites being selectively accumulated in different tissues.

Topics: Animals; beta Carotene; Chromatography, High Pressure Liquid; Female; Gonads; Isomerism; Male; Molecular Structure; Muscles; Sexual Maturation; Skin; Skin Pigmentation; Trout; Xanthophylls

The freshwater microalga Haematococcus pluvialis is one of the best microbial sources of the carotenoid astaxanthin, but this microalga shows low growth rates and low final cell densities when cultured with traditional media. A single-variable optimization strategy was applied to 18 components of the culture media in order to maximize the productivity of vegetative cells of H. pluvialis in semicontinuous culture. The steady-state cell density obtained with the optimized culture medium at a daily volume exchange of 20% was 3.77 x 10(5) cells ml(-1), three times higher than the cell density obtained with Bold basal medium and with the initial formulation. The formulation of the optimal Haematococcus medium (OHM) is (in g l(-1)) KNO3 0.41, Na2HPO4 0.03, MgSO4 x 7H2O 0.246, CaCl2 x 2H2O 0.11, (in mg l(-1)) Fe(III)citrate x H2O 2.62, CoCl2 x 6H2O 0.011, CuSO4 x 5H2O 0.012, Cr2O3 0.075, MnCl2 x 4H2O 0.98, Na2MoO4 x 2H2O 0.12, SeO2 0.005 and (in microg l(-1)]) biotin 25, thiamine 17.5 and B12 15. Vanadium, iodine, boron and zinc were demonstrated to be non-essential for the growth of H. pluvialis. Higher steady-state cell densities were obtained by a three-fold increase of all nutrient concentrations but a high nitrate concentration remained in the culture medium under such conditions. The high cell productivities obtained with the new optimized medium can serve as a basis for the development of a two-stage technology for the production of astaxanthin from H. pluvialis.

Topics: beta Carotene; Chlorophyta; Culture Media; Light; Nitrogen; Xanthophylls

Duplicate groups of Atlantic salmon post-smolts were fed four purified diets supplemented with both vitamin E and the carotenoid astaxanthin (Ax) (+E, +Ax), or supplemented with either vitamin E or Ax (-E, +Ax and +E, -Ax) or deficient in both vitamin E and Ax (-E, -Ax) for 22 wk. There were no effects of diet on growth rate, but an extensive lipoid liver degenerative lesion was observed in 15% of fish fed diets deficient in vitamin E. Tissue vitamin E concentrations varied in accordance with dietary vitamin E in liver, muscle, heart, plasma, brain and eye; levels were reduced to approximately 3% in liver but only to 40% in eye of fish fed diets deficient in vitamin E compared with those fed diets supplemented with vitamin E. An interactive sparing of Ax supplementation on tissue vitamin E concentration was observed, but only in brain. Dietary deficiency of both vitamin E and Ax significantly increased the recovery of desaturated and elongated products of both [1-(14)C] 18:3(n-3) and [1-(14)C] 20:5(n-3) in isolated hepatocytes, suggesting that conversion of fatty acids to their long-chain highly unsaturated products can be stimulated by a deficiency of lipid-soluble antioxidants. The antioxidant synergism of vitamin E and Ax was supported by their ability to reduce malondialdehyde formation in an in vitro stimulation of microsomal lipid peroxidation and to reduce plasma levels of 8-isoprostane. The results of this study suggest that both vitamin E and the carotenoid Ax have antioxidant functions in Atlantic salmon.

Topics: Animal Feed; Animals; Antioxidants; Aquaculture; beta Carotene; Dietary Supplements; Dinoprost; F2-Isoprostanes; Fatty Acids; Oxidation-Reduction; Salmo salar; Vitamin E; Weight Gain; Xanthophylls

Topics: Amines; beta Carotene; Canthaxanthin; Dimyristoylphosphatidylcholine; Free Radical Scavengers; Kinetics; Liposomes; Molecular Conformation; Organophosphates; Oxygen; Phospholipids; Photochemistry; Singlet Oxygen; Vitamin E; Xanthophylls

Using metabolic engineering, we have modified the carotenoid biosynthesis pathway in tobacco (Nicotiana tabacum) to produce astaxanthin, a red pigment of considerable economic value. To alter the carotenoid pathway in chromoplasts of higher plants, the cDNA of the gene CrtO from the alga Haematococcus pluvialis, encoding beta-carotene ketolase, was transferred to tobacco under the regulation of the tomato Pds (phytoene desaturase) promoter. The transit peptide of PDS from tomato was used to target the CRTO polypeptide to the plastids. Chromoplasts in the nectary tissue of transgenic plants accumulated (3S,3'S) astaxanthin and other ketocarotenoids, changing the color of the nectary from yellow to red. This accomplishment demonstrates that plants can be used as a source of novel carotenoid pigments such as astaxanthin. The procedures described in this work can serve as a platform technology for future genetic manipulations of pigmentation of fruits and flowers of horticultural and floricultural importance.

Topics: Bacterial Proteins; beta Carotene; DNA, Complementary; Nicotiana; Oxygenases; Plants, Genetically Modified; Plants, Toxic; Xanthophylls

The effect of different oils on the absorption of carotenoids was investigated in mesenteric lymph duct cannulated rats. Sixteen treatment emulsions containing increasing concentrations of either lycopene (LYC) or astaxanthin (AST) (5, 10, 15, 20 micromol/L) were prepared with olive oil or corn oil and continuously infused into the duodenum of the rat. Absorption of carotenoids into the mesenteric lymph duct was determined. Absorption of LYC and AST from both oils increased with the amount infused into the duodenum. The average recovery of AST in the lymph from the olive oil emulsion was 20% but was decreased to 13% from emulsions containing corn oil. Lycopene was not as well absorbed as AST. The average recovery of LYC was 6% from olive oil emulsions but only 2.5% when infused with corn oil. The LYC used in this study was isolated from tomato paste and was primarily in the all-trans form. We did not observe any significant isomerization of all-trans LYC to 9-cis LYC during absorption. We conclude that the type of oil with which a carotenoid is consumed can influence its absorption.

Topics: Animals; beta Carotene; Carotenoids; Corn Oil; Dietary Fats; Duodenum; Emulsions; Infusions, Parenteral; Intestinal Absorption; Lycopene; Lymph; Male; Olive Oil; Plant Oils; Rats; Rats, Sprague-Dawley; Xanthophylls

Helicobacter pylori infection in humans is associated with chronic type B gastritis, peptic ulcer disease, and gastric carcinoma. A high intake of carotenoids and vitamin C has been proposed to prevent development of gastric malignancies. The aim of this study was to explore if the microalga Haematococcus pluvialis rich in the carotenoid astaxanthin and vitamin C can inhibit experimental H. pylori infection in a BALB/cA mouse model. Six-week-old BALB/cA mice were infected with the mouse-passaged H. pylori strain 119/95. At 2 weeks postinoculation mice were treated orally once daily for 10 days (i) with different doses of algal meal rich in astaxanthin (0.4, 2, and 4 g/kg of body weight, with the astaxanthin content at 10, 50, and 100 mg/kg, respectively), (ii) with a control meal (algal meal without astaxanthin, 4 g/kg), or (iii) with vitamin C (400 mg/kg). Five mice from each group were sacrificed 1 day after the cessation of treatment, and the other five animals were sacrificed 10 days after the cessation of treatment. Culture of H. pylori and determination of the inflammation score of the gastric mucosae were used to determine the outcome of the treatment. Mice treated with astaxanthin-rich algal meal or vitamin C showed significantly lower colonization levels and lower inflammation scores than those of untreated or control-meal-treated animals at 1 day and 10 days after the cessation of treatment. Lipid peroxidation was significantly decreased in mice treated with the astaxanthin-rich algal meal and vitamin C compared with that of animals not treated or treated with the control meal. Both astaxanthin-rich algal meal and vitamin C showed an inhibitory effect on H. pylori growth in vitro. In conclusion, antioxidants may be a new strategy for treating H. pylori infection in humans.

Topics: Agar; Animals; Ascorbic Acid; beta Carotene; Carotenoids; Disease Models, Animal; Helicobacter Infections; Helicobacter pylori; Lipid Peroxidation; Mice; Mice, Inbred BALB C; Xanthophylls

We studied the carotenoid pigments in plasma, skin and body fat of white storks (Ciconia ciconia) from a colony in Spain feeding mainly on the recently introduced red swamp crayfish (Procambarus clarkii). In control colonies, where crayfish was absent, plasma was collected for comparison. Our objective was to determine whether the astaxanthin contained in the crayfish reached the blood, accumulated in fat, and finally was deposited in the red-colored bill and legs. If that was true, the visual cues provided by those tegumentary areas would be altered, with potential behavioral consequences. Plasma carotenoids were directly extracted with acetone, whereas skin and fat samples needed harsher conditions, i.e. grinding, sonication and extraction with diethyl ether. The extracts were analyzed by thin-layer chromatography (TLC) and UV/Vis spectroscopy. In crayfish-eating storks, astaxanthin was confirmed to be the dominant pigment in all analyzed tissues. This red pigment was absorbed unchanged in the gut, and was responsible for the red color of plasma and the abnormal orange pigmentation of the feather-covered skin. It was also present in large quantities in the exposed bill and tarsi, which are typically red-colored in the stork. Control storks with no crayfish in the diet only presented lutein in their plasma.

Topics: Animals; Astacoidea; beta Carotene; Birds; Diet; Pigmentation; Xanthophylls

Intracellular production of active oxygen in the green alga Haematococcus pluvialis was studied by measuring the capacity for in vivo conversion of 2',7'-dichlorohydrofluorescein diacetate to the fluorescent dye dichlorofluorescein in different algal cell types (i.e., vegetative, immature cyst and mature cyst cells). The increase in formation of dichlorofluorescein by methyl viologen (superoxide anion radical generator) was linear for 2 h in immature cyst cells (low astaxanthin) in a methyl viologen-concentration-dependent manner, while no production was detected in mature (high astaxanthin) cysts. Compared to cyst cells, formation of dichlorofluorescein in vegetative cells (no astaxanthin) was markedly increased by methyl viologen. The formation of dichlorofluorescein in cyst cells was decreased with higher astaxanthin content under excessive oxidative stress. All of the active oxygen species tested (singlet oxygen, superoxide anion radical, hydrogen peroxide and peroxy radical) at 10(-3) M increased the intracellular dichlorofluorescein formation in immature cysts, but did not increase the dichlorofluorescein content of mature cysts. Therefore, astaxanthin in cyst cells appeared to function as an antioxidant agent against oxidative stress.

Topics: Antioxidants; beta Carotene; Chlorophyta; Fluoresceins; Oxidative Stress; Xanthophylls

The developing eggs of the crayfish Astacus leptodactylus are found to contain important carotenoid amounts. Free astaxanthin, mainly associated with protein and lutein represent the main pigments occurring in the yolk at the end of the embryonic period. Esterification processes affecting astaxanthin appear concomitantly with the resorption of vitellin reserves, the activation of the digestive gland and the rapid decrease of lutein amounts. Esterified astaxanthin contents increase significantly within the interval between the juvenile stages I and II. Nevertheless, free astaxanthin levels appear insufficient to balance the amounts of the esterified form, scored at the end of the endotrophic period.

Topics: Animals; Astacoidea; beta Carotene; Lutein; Xanthophylls; Zeaxanthins

Apparent astaxanthin (3,3'-dihydroxy-beta,beta-carotene-4,4'-dione) digestibility coefficients (ADC) and carotenoid compositions of the muscle, liver, whole kidney and plasma were compared in Atlantic salmon (Salmo salar) and Atlantic halibut (Hippoglossus hippoglossus) fed a diet supplemented with 66 mg astaxanthin kg(-1) dry matter for 112 days. The astaxanthin source consisted of 75% all-E-, 3% 9Z- and 22% 13Z-astaxanthin, of (3R,3'R)-, (3R,3'S; meso)-, and (3S,3'S)-astaxanthin in a 1:2:1 ratio. The ADC of astaxanthin was significantly higher in Atlantic halibut than in Atlantic salmon after 56 and 112 days of feeding (P < 0.05). The ADC of all-E-astaxanthin was significantly higher than ADC of 9Z-astaxanthin (P < 0.05). Considerably more carotenoids were present in all plasma and tissue samples of salmon than in halibut. Retention of astaxanthin in salmon muscle was 3.9% in salmon and 0 in halibut. All-E-astaxanthin accumulated selectively in the muscle of salmon, and in plasma of salmon and halibut compared with diet. 13Z-astaxanthin accumulated selectively in liver and whole kidney of salmon and halibut, when compared with plasma. A reductive pathway for astaxanthin metabolism in halibut similar to that of salmon was shown by the presence of 3',4'-cis and trans glycolic isomers of idoxanthin (3,3',4'-trihydroxy-beta,beta-carotene-4'-one) in plasma, liver and whole kidney. In conclusion, the higher ADC of astaxanthin in halibut than Atlantic salmon may be explained by lower feed intake in halibut, and the lower retention of astaxanthin by a higher capacity to transform astaxanthin metabolically.

Topics: Animals; Atlantic Ocean; beta Carotene; Carotenoids; Fishes; Isomerism; Molecular Structure; Salmo salar; Xanthophylls

Marine animals produce astaxanthin which is a carotenoid and antioxidant. In this study we determined the in vitro and ex vivo effects of astaxanthin on LDL oxidation. The oxidation of LDL was measured in a 1 ml reaction system consisting of increasing concentrations of astaxanthin (12.5, 25.0, 50.0 microg/ml), 400 microM V-70 (2, 2'-azobis(4-methoxy-2, 4-dimethylvaleronitrile)), and LDL (70 microg/ml protein). Astaxanthin dose, dependently significantly prolonged the oxidation lag time (31.5, 45.4, 65.0 min) compared with the control (19.9 min). For the ex vivo study 24 volunteers (mean age 28.2 [SD 7.8] years) consumed astaxanthin at doses of 1.8, 3.6,14.4 and 21.6 mg per day for 14 days. No other changes were made in the diet. Fasting venous blood samples were taken at days 0, +14. LDL lag time was longer (5.0, 26.2, 42.3 and 30.7% respectively) compared with day 0 after consuming astaxanthin at doses of 1.8, 3.6,14.4 and 21.6 mg for 14 days compared with day 0, but there was no difference in oxidation of LDL between day 0 (lag time 59.9+/-7.2 min) and day 14 (57.2+/-6.0 min) in the control group. Our results provide evidence that consumption of marine animals producing astaxanthin inhibits LDL oxidation and possibly therefore contributes to the prevention of atherosclerosis.

Topics: Adult; Animals; Antioxidants; Apoproteins; beta Carotene; Case-Control Studies; Crustacea; Dose-Response Relationship, Drug; Humans; In Vitro Techniques; Lipids; Lipoproteins; Lipoproteins, LDL; Oxidation-Reduction; Xanthophylls

The antioxidative biological effect of dietary red yeast, Phaffia rhodozyma, which is rich in astaxanthin, on rainbow trout, Oncorhynchus mykiss, was examined. The levels of serum transaminase (glutamic-pyruvic transaminase and glutamic-oxaloacetic transaminase) activities and of lipid peroxides (LPO) of fish fed oxidized oil were significantly higher than those of the control fish fed non-oxidized oil. However, the supply of red yeast considerably decreased both enzyme activities and LPO level. Furthermore, the serum lipid (triglycerides, total cholesterol and phospholipids) concentrations were also significantly decreased. Especially, the serum triglyceride level of fish fed the red yeast was as low as that of the control. It was also observed that there were no significant differences in muscle LPO levels between the fish fed red yeast and the control. The present results suggest for the first time that dietary red yeast may effectively suppress the LPO generation of tissue and normalize liver function as well as improving muscle pigmentation of trout. Thus, red yeast should have a reducing effect on oxidized oil-induced oxidative stress in fish.

Topics: Animal Feed; Animals; Antioxidants; beta Carotene; Lipid Peroxides; Muscles; Oncorhynchus mykiss; Oxidative Stress; Thiobarbituric Acid Reactive Substances; Triglycerides; Xanthophylls; Yeasts

Concentrations of all-E-, 9Z- and 13Z- geometrical and (3R,3'R), (3R, 3'S) and (3S,3'S) optical isomers of astaxanthin were determined in rainbow trout liver, gut tissues, kidney, skin and blood plasma to evaluate their body distribution. Two cold-pelleted diets containing predominantly all-E-astaxanthin (36.9 mg/kg astaxanthin, 97% all-E-, 0.4% 9Z-, 1.5% 13Z-astaxanthin, and 1.1% other isomers, respectively) or a mixture of all-E- and Z-astaxanthins (35.4 mg/kg astaxanthin, 64% all-E-, 18.7% 9Z-, 12.3% 13Z-astaxanthin, and 2.0% other isomers, respectively), were fed to duplicate groups of trout for 69 d. Individual E/Z isomers were identified by VIS- and 1H-NMR-spectrometry, and quantified by high-performance liquid chromatography. Significantly higher total carotenoid concentration was observed in plasma of trout fed diets with all-E-astaxanthin (P < 0.05). The relative E/Z-isomer concentrations of plasma, skin and kidney were not significantly different among groups, whereas all-E-astaxanthin was higher in intestinal tissues and 13Z-astaxanthin was lower in liver of trout fed all-E-astaxanthin (P < 0.05). The relative amount of hepatic 13Z-astaxanthin (39-49% of total astaxanthin) was higher than in all other samples (P < 0.05). Synthetic, optically inactive astaxanthin was used in all experiments, and the determined dietary ratio between the 3R,3'R:3R, 3'S (meso):3S,3'S optical isomers was 25.3:49.6:25.1. The distribution of R/S-astaxanthin isomers in feces, blood, liver and fillet was similar to that in the diets. The ratio between (3S,3'S)- and (3R,3'R)-astaxanthin in the skin and posterior kidney was ca. 2:1 and 3:1, respectively, regardless of dietary E/Z-astaxanthin composition. The results show that geometrical and optical isomers of astaxanthin are distributed selectively in different tissues of rainbow trout.

Topics: Animals; beta Carotene; Carotenoids; Chromatography, High Pressure Liquid; Feces; Intestinal Mucosa; Kidney; Liver; Magnetic Resonance Spectroscopy; Metabolic Clearance Rate; Oncorhynchus mykiss; Skin; Spectrophotometry; Stereoisomerism; Tissue Distribution; Xanthophylls

The strain E-396T, isolated from soil, was Gram-negative, aerobic, orange-pigmented, rod-shaped, motile by peritrichous flagella and astaxanthin-producing. This organism produced carotenoids, mainly astaxanthin, and did not produce bacteriochlorophyll. The ubiquinone system was Q-10. Analysis of the 16S rRNA sequence of strain E-396T showed it to be a member of the alpha-3 subclass of the Proteobacteria, forming a cluster with the species of the genus Paracoccus. On the basis of the production of orange pigments and motility by peritrichous flagella, together with DNA-DNA reassociation data, it is concluded that the new isolate should be classified into a new species of the genus Paracoccus, Paracoccus carotinifaciens sp. nov. The type strain is E-396T (= IFO 16121T).

Topics: Base Sequence; beta Carotene; DNA, Bacterial; Molecular Sequence Data; Paracoccus; Phylogeny; RNA, Ribosomal, 16S; Xanthophylls

The subunit C1 of the carotenoid-binding protein, V600, of the chondrophore Velella velella has been purified and crystallized. The crystals, which were grown by the vapour-diffusion method from ammonium sulfate as the major precipitant, diffract beyond 3 A and show little radiation damage over long periods (greater than 100 h) on a Cu Kalpha rotating-anode X-ray source. The space group of the crystals is P212121 with cell dimensions a = 42.0, b = 80.9, c = 110. 6 A.

Topics: Animals; beta Carotene; Crystallization; Crystallography, X-Ray; Hydra; Protein Conformation; Xanthophylls

The isoprenoid pathway is a versatile biosynthetic network leading to over 23,000 compounds. Similar to other biosynthetic pathways, the production of isoprenoids in microorganisms is controlled by the supply of precursors, among other factors. To engineer a host that has the capability to supply geranylgeranyl diphosphate (GGPP), a common precursor of isoprenoids, we cloned and overexpressed isopentenyl diphosphate (IPP) isomerase (encoded by idi) from Escherichia coli and GGPP synthase (encoded by gps) from the archaebacterium Archaeoglobus fulgidus. The latter was shown to be a multifunctional enzyme converting dimethylallyl diphosphate (DMAPP) to GGPP. These two genes and the gene cluster (crtBIYZW) of the marine bacterium Agrobacterium aurantiacum were introduced into E. coli to produce astaxanthin, an orange pigment and antioxidant. This metabolically engineered strain produces astaxanthin 50 times higher than values reported before. To determine the rate-controlling steps in GGPP production, the IDI-GPS pathway was compared with another construct containing idi, ispA (encoding farnesyl diphosphate (FPP) synthase in E. coli), and crtE (encoding GGPP synthase from Erwinia uredovora). Results show that the conversion from FPP to GGPP is the first bottleneck, followed sequentially by IPP isomerization and FPP synthesis. Removal of these bottlenecks results in an E. coli strain providing sufficient precursors for in vivo synthesis of isoprenoids.

Topics: Alkyl and Aryl Transferases; Archaea; Base Sequence; beta Carotene; Biotechnology; Carbon-Carbon Double Bond Isomerases; Cloning, Molecular; DNA Primers; Escherichia coli; Farnesyltranstransferase; Gene Expression; Genetic Engineering; Hemiterpenes; Kinetics; Polyisoprenyl Phosphates; Xanthophylls

The metabolism of the nonprovitamin A carotenoid astaxanthin was investigated in primary cultures of rat hepatocytes. In a time course study based on HPLC and gas chromatography-mass spectrometry analyses, one main metabolite, (rac)-3-hydroxy-4-oxo-beta-ionone, was found. This metabolite was conjugated mainly into glucuronides, as demonstrated by glusulase treatment of the conjugates under sulfatase-inhibiting conditions. Within 24 h more than 50% astaxanthin was metabolized and conjugated. Deconjugation of the polar conjugates with glusulase and analyses with HPLC and gas chromatography-mass spectrometry identified two metabolites, (rac)-3-hydroxy-4-oxo-beta-ionone and its reduced form (rac)-3-hydroxy-4-oxo-7,8-dihydro-beta-ionone, indicating that the former was reduced in the conjugated form. We confirmed that the ketocarotenoid astaxanthin induces xenobiotic-metabolizing enzymes in rat liver in vivo. However, there were no differences in the metabolism of astaxanthin in cultured hepatocytes from rats that were pretreated with astaxanthin and, thus, with induced cytochrome P-450 systems compared with control hepatocytes. Neither liver microsomes from astaxanthin-pretreated nor control rats metabolized astaxanthin. These results indicated that the cytochrome P-450 enzymes were not involved in the metabolism of astaxanthin in rat hepatocytes. We conclude that astaxanthin was metabolized in primary cultures of rat hepatocytes into (rac)-3-hydroxy-4-oxo-beta-ionone and its reduced form (rac)-3-hydroxy-4-oxo-7,8-dihydro-beta-ionone independent of the xenobiotic-metabolizing enzymes induced by astaxanthin.

Topics: Animals; beta Carotene; Carbon Radioisotopes; Cells, Cultured; Chromatography, High Pressure Liquid; Cytochrome P-450 CYP1A1; Gas Chromatography-Mass Spectrometry; Male; Microsomes, Liver; Rats; Rats, Wistar; Xanthophylls; Xenobiotics

Semiempirical AM1 calculations have been carried out for beta-carotene and the three xanthophylls (zeaxanthin, canthaxanthin, and astaxanthin) containing oxygen functions (hydroxy/keto groups) found in the majority of natural pigment. The fully optimized geometries correspond well with the X-ray structures of beta-carotene and canthaxanthin and indicate that substitutions on the terminal rings have a minimal effect on the conformation of the chromophore. Twisting along the polyenic chain results from steric interaction involving methyl substituents, and a Ci point group can be proposed for the four investigated carotenoids. AM1 calculated excitation energies for the strongly allowed excited states can be compared to with the experimental absorption band in the visible region, considering solvent effect. Resonance Raman (RR) and Fourier transform (FT) Raman spectra of natural astaxanthin as well as astaxanthins specifically 13C labeled at the positions 12,12'; 13,13'; 14,14'; 15,15'; 15, and 20,20' were recorded. Furthermore the RR and FT Raman spectra of the asymmetric carotenoid 20-norastaxanthin are presented. The data reveal a substantial amount of information about the coupling between the different vibrations, and enabled an extensive experimental verification of the theoretical normal-coordinate analysis previously performed on polyenic molecules [J Raman Spectrosc 1983, 14, 310-321; Advances in Infrared and Raman Spectroscopy, Vol. 12, 1985, pp. 115-178; Spectrochim Acta 1996, 53, 381-392; Biochim Biophys Acta 1994, 1185, 188-196]. The results make up a very interesting dataset which allowed the interpretation and/or observation of several, hitherto never observed or not well understood, effects in the Raman spectra of the differently labeled astaxanthins.

Topics: Absorption; beta Carotene; Canthaxanthin; Carotenoids; Electrons; Lasers; Models, Chemical; Models, Molecular; Molecular Conformation; Molecular Structure; Normal Distribution; Spectroscopy, Fourier Transform Infrared; Spectrum Analysis, Raman; Xanthophylls; Zeaxanthins

This study investigates the effect of dietary carotenoids on pim-1 gene expression in mouse splenocytes. Female BALB/c mice were fed 0%, 0.02%, or 0.4% astaxanthin, beta-carotene, and lutein for two weeks. Plasma and liver were obtained for the analysis of carotenoids. Splenocytes were isolated and cultured in the presence of concanavalin A, and the level of pim-1 mRNA was determined by Northern blot analysis. None of the carotenoids were detectable in the plasma and liver of unsupplemented mice. In plasma the concentration of astaxanthin (4.9-54.7 mumol/l) was dramatically higher than that of lutein (1.4-2.0 mumol/l) and beta-carotene (0.1-0.7 mumol/l). Carotenoid uptake by the spleen but not the liver reflected that observed in plasma. In mice fed 0.4% of each carotenoid, the absolute concentration of the carotenoid in the liver was highest for astaxanthin (24 nmol/g) followed by beta-carotene (7.5 nmol/g) and lutein (1.58 nmol/g). Mice fed lutein showed a dose-related increase in pim-1 mRNA expression. The steady-state level of pim-1 mRNA in mice fed 0.4% lutein was sixfold higher than in mice fed 0.02% lutein. In contrast, dietary astaxanthin and beta-carotene did not affect pim-1 expression. Therefore, an increase in pim-1 mRNA was observed in splenocytes stimulated with concanavalin A in lutein-fed mice. This appears to be a unique effect of lutein and may be associated with its antitumor activity observed in vivo.

Topics: Animals; Anticarcinogenic Agents; beta Carotene; Blotting, Northern; Chromatography, High Pressure Liquid; Diet; Female; Gene Expression Regulation; Liver; Lutein; Lymphocytes; Mice; Mice, Inbred BALB C; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-pim-1; RNA, Messenger; Spleen; Xanthophylls

The first carotenoid biosynthetic gene from the basidiomycetous yeast Xanthophyllomyces dendrorhous was isolated by heterologous complementation in Escherichia coli. The isolated gene, denominated as crtI, was found to encode for phytoene desaturase. The coding region is interrupted by 11 introns. The deduced amino acid sequence showed significant homology with its bacterial and eukaryotic counterparts, especially those of fungal origin. A plasmid containing the geranylgeranyl diphosphate synthase and phytoene synthase encoding genes from Erwinia uredovora was introduced in E. coli together with the phytoene desaturase encoding cDNA from X. dendrorhous. As a result, lycopene accumulation was observed in these transformants. We conclude that in X. dendrorhous the four desaturase steps, by which phytoene is converted into lycopene, are carried out by a single gene product.

Topics: Amino Acid Sequence; Base Sequence; Basidiomycota; beta Carotene; Carotenoids; Cloning, Molecular; Escherichia coli; Fungal Proteins; Genetic Complementation Test; Lycopene; Molecular Sequence Data; Oxidoreductases; Recombinant Proteins; Sequence Analysis, DNA; Xanthophylls

The effect of 16 d intake of 300 mg carotenoids/kg diet (beta-carotene (beta C), bixin (BX), lycopene (LY), lutein (LU), canthaxanthin (CX) or astaxanthin (AX) on xenobiotic metabolizing enzymes in the liver, lung, kidney and small intestine of male Wistar rats was assessed. A control group received the basal diet (AIN-76) without carotenoids and a positive control group for enzyme induction received 3-methylcholanthrene (3-MC) at 666 mg/kg diet. Cytochrome P450 activity was assessed using the substrates ethoxyresorufin for P450 1A1, methoxyresorufin for P450 1A2, pentoxyresorufin for P450 2B1/2 and benzyloxyresorufin for P450 types 1A1/2, 2B1/2 and 3A. Glutathione-S-transferase (EC 2.5.1.18) and reduced glutathione status were assessed. Carotenoid uptake by the tissues was also determined. 3-MC and the carotenoids BX, CX and AX led to significant increases compared with control in liver, lung and kidney ethoxyresorufin-O-deethylation. Methoxyresorufin-O-demethylation activity was significantly increased in liver and lung by BX, CX and AX but only CX and AX significantly increased activity in kidney. Pentoxyresorufin-O-depentylation and benzyloxyresorufin-O-dearylation increased in liver of 3-MC-, BX-, CX- and AX-treated rats, but to a much lesser degree than for the other two substrates. Benzyloxyresorufin-O-dearylation in lung was significantly decreased by all carotenoids. Activities of any of the measured enzymes in the small intestine were undetectable in all treatment groups except the 3-MC group. Glutathione status was unaffected by any of the treatments. This is the first study identifying the carotenoids BX, CX and AX as inducers of rat lung and kidney xenobiotic metabolizing enzymes.

Topics: Animals; Anticarcinogenic Agents; beta Carotene; Canthaxanthin; Carcinogens; Carotenoids; Cytochrome P-450 Enzyme System; Enzyme Activation; Glutathione; Glutathione Transferase; Intestine, Small; Kidney; Liver; Lung; Lutein; Lycopene; Male; Methylcholanthrene; Rats; Rats, Wistar; Xanthophylls; Xenobiotics

High cell density cultivation of Haematococcus pluvialis for astaxanthin production was carried out in batch and fed-batch modes in 3.7-L bioreactors with stepwise increased light intensity control mode. A high cell density of 2.65 g x L(-1) (batch culture) or 2.74 g x L(-1) (fed-batch culture) was obtained, and total astaxanthin production in the fed-batch culture (64.36 mg x L(-1)) was about 20.5% higher than in the batch culture (53.43 mg x L(-1)). An unstructured kinetic model to describe the microalga culture system including cell growth, astaxanthin formation, as well as sodium acetate consumption was proposed. Good agreement was found between the model predictions and experimental data. The models demonstrated that the optimal light intensity for mixotrophic growth of H. pluvialis in batch or fed-batch cultures in a 3.7-L bioreactor was 90-360 micromol x m(-2) x s(-1), and that the stepwise increased light intensity mode could be replaced by a constant light intensity mode.

Topics: Animals; beta Carotene; Bioreactors; Cell Culture Techniques; Chlorophyta; Computer Simulation; Kinetics; Light; Models, Biological; Sodium Acetate; Xanthophylls

The reproducibility of the separation of astaxanthin stereoisomers on columns packed with Pirkle covalent L-leucine chiral stationary phase (CSP) was examined by comparing six columns purchased from the same manufacturer. Differences were found even for columns packed with CSP from the same lot. The reproducibility of columns packed with Pirkle covalent D-phenylglycine CSP was also examined by comparing columns purchased from the same manufacturer as well as from different manufacturers. Significant differences were found for columns packed by different manufacturers. Chiral column-to-column reproducibility for complex stereoisomeric separations should therefore not be taken for granted.

Topics: beta Carotene; Chromatography, High Pressure Liquid; Glycine; Leucine; Reproducibility of Results; Spectrophotometry, Ultraviolet; Stereoisomerism; Xanthophylls

A cDNA homologous to beta-carotene hydroxylase from Arabidopsis thaliana was isolated from the green alga Haematococcus pluvialis. The predicted amino acid sequence for this enzyme shows homology to the three known plant beta-carotene hydroxylases from Arabidopsis thaliana and from Capsicum annuum (38% identity) and to prokaryote carotenoid hydroxylases (32-34% identities). Heterologous complementation using E. coli strains which were genetically engineered to produce carotenoids indicated that the H. pluvialis beta-carotene hydroxylase was able to catalyse not only the conversion of beta-carotene to zeaxanthin but also the conversion of canthaxanthin to astaxanthin. Furthermore, Northern blot analysis revealed increased beta-carotene hydroxylase mRNA steady state levels after induction of astaxanthin biosynthesis. In accordance with the latter results, it is proposed that the carotenoid hydroxylase characterized in the present publication is involved in the biosynthesis of astaxanthin during cyst cell formation of H. pluvialis.

Topics: Amino Acid Sequence; Base Sequence; beta Carotene; Canthaxanthin; Chlorophyta; Conserved Sequence; DNA, Complementary; Escherichia coli; Gene Expression Regulation; Gene Library; Mixed Function Oxygenases; Molecular Sequence Data; Plasmids; RNA, Messenger; Xanthophylls

Two practical methods are reported for treating feral Baltic salmon with thiamine hydrochloride against M74 syndrome (abnormally high yolk-sac fry mortality of the Baltic salmon). Both bathing of the yolk-sac fry in thiamine hydrochloride (1000 mg l-1, 1 h) and a single intraperitoneal injection given to the female brood fish (100 mg kg-1 fish) during the summer 3 mo before stripping were shown to elevate the whole body total thiamine concentration in the fry. Both treatments were also shown to be effective in preventing mortality due to M74 syndrome. The effect of bathing the yolk-sac fry was shown to be dose-dependent. The results support the view that there is a causal relationship between the thiamine status of the yolk-sac fry and M74 mortality. An intraperitoneal injection of astaxanthine suspension administered to the female brood fish (11 mg kg-1 fish) in the summer 3 mo before stripping elevated the astaxanthine concentration in the eggs but did not affect mortality due to M74 syndrome. An interaction between astaxanthine and thiamine may occur in the developing embryo or yolk-sac fry, however. No association could be demonstrated between the various thiamine hydrochloride treatment practices and hepatic cytochrome P450 dependent 7-ethoxyresorufin-O-deethylase (EROD) activity in the yolk-sac fry. An injection of thiamine hydrochloride into the peritoneal cavity of wild Baltic salmon females could be used to raise thiamine concentrations in their offspring in the rivers. The effect on smolt production in Finnish Baltic salmon rivers needs to be investigated further, however.

Topics: Adjuvants, Immunologic; Administration, Topical; Animals; beta Carotene; Chromatography, High Pressure Liquid; Cytochrome P-450 CYP1A1; Dose-Response Relationship, Drug; Female; Fish Diseases; Injections, Intraperitoneal; Muscle, Skeletal; Random Allocation; Salmo salar; Syndrome; Thiamine; Xanthophylls; Yolk Sac

The isomerization of trans-astaxanthin to cis-isomers in organic solvents was investigated. trans-Astaxanthin was dissolved in dimethyl sulfoxide, dichloromethane, chloroform, acetone, methanol, acetonitrile, and a mixture of dichloromethane and methanol (25:75) respectively, and heated at 35 degrees C followed by analyzing cis- and trans-astaxanthins in the solutions using HPLC. The isomerization rates of trans-astaxanthin were dependent on the solvent, and the following order was found: dichloromethane > chloroform > the mixture of dichloromethane and methanol (25:75) > methanol > acetonitrile > acetone > dimethyl sulfoxide. In different solvents, the relative contents of 9-cis- and 13-cis-astaxanthins formed during isomerization were different. In all solvents, 13-cis-isomer was the main cis-isomer from trans-astaxanthin. trans-Astaxanthin dissolved in dichloromethane or chloroform was very readily isomerized to cis-isomers, especially for dichloromethane, in which a maximum isomerization percentage was found and an equilibrium practically was reached after an appropriate time interval. Results also indicated that a higher temperature could promote markedly the isomerization of trans-astaxanthin.

Topics: beta Carotene; Kinetics; Models, Molecular; Solvents; Spectrophotometry; Stereoisomerism; Xanthophylls

The reaction kinetics for the hydrolysis of astaxanthin esters and the degradation of astaxanthin during saponification of the pigment extract from the microalga Haematococcus pluvialis were investigated. Different concentrations of sodium hydroxide in methanol were used for the saponification under nitrogen in darkness at ambient temperature (22 degrees C) followed by the analysis of astaxanthins and other carotenoids using an HPLC method. The concentration of methanolic NaOH solution was important for promoting the hydrolysis of astaxanthin esters and minimizing the degradation of astaxanthin during saponification. With a higher concentration of methanolic NaOH solution, the reaction rate of hydrolysis was high, but the degradation of astaxanthin occurred significantly. The rate constants of the hydrolysis reaction (first order) of astaxanthin esters and the degradation reaction (zero-order) of astaxanthin were directly proportional to the concentration of sodium hydroxide in the saponified solution. Although the concentration of sodium hydroxide in the saponified solution was 0.018 M, complete hydrolysis of astaxanthin esters was achieved in 6 h for different concentrations (10-100 mg/L) of pigment extracts. Results also indicated that a higher temperature should be avoided to minimize the degradation of astaxanthin. In addition, during saponification, no loss of lutein, beta-carotene, and canthaxanthin was found.

Topics: beta Carotene; Chromatography, High Pressure Liquid; Esters; Hydrolysis; Hydroxides; Kinetics; Potassium Compounds; Sodium Hydroxide; Temperature; Xanthophylls

The red heterobasidiomycetous yeast Xanthophyllomyces dendrorhous (perfect state of Phaffia rhodozyma) contains a novel type of carotenoid biosynthetic enzyme. Its structural gene, designated crtYB, was isolated by functional complementation in a genetically modified, carotenogenic Escherichia coli strain. Expression studies in different carotenogenic E. coli strains demonstrated that the crt YB gene encodes a bifunctional protein involved both in synthesis of phytoene from geranylgeranyl diphosphate and in cyclisation of lycopene to beta-carotene. By sequence comparison with other phytoene synthases and complementation studies in E. coli with various deletion derivatives of the crtYB gene, the regions responsible for phytoene synthesis and lycopene cyclisation were localised within the protein.

Topics: Alkyl and Aryl Transferases; Amino Acid Sequence; Basidiomycota; beta Carotene; Carotenoids; Cloning, Molecular; DNA, Complementary; Genes, Fungal; Geranylgeranyl-Diphosphate Geranylgeranyltransferase; Intramolecular Lyases; Molecular Sequence Data; Peptide Fragments; Protein Structure, Tertiary; Sequence Homology, Amino Acid; Xanthophylls; Yeasts

Resonance Raman spectroscopy and quantum chemical calculations were used to investigate the molecular origin of the large redshift assumed by the electronic absorption spectrum of astaxanthin in alpha-crustacyanin, the major blue carotenoprotein from the carapace of the lobster, Homarus gammarus. Resonance Raman spectra of alpha-crustacyanin reconstituted with specifically 13C-labeled astaxanthins at the positions 15, 15,15', 14,14', 13,13', 12,12', or 20,20' were recorded. This approach enabled us to obtain information about the effect of the ligand-protein interactions on the geometry of the astaxanthin chromophore in the ground electronic state. The magnitude of the downshifts of the C==C stretching modes for each labeled compound indicate that the main perturbation on the central part of the polyene chain is not homogeneous. In addition, changes in the 1250-1400 cm(-1) spectral range indicate that the geometry of the astaxanthin polyene chain is moderately changed upon binding to the protein. Semiempirical quantum chemical modeling studies (Austin method 1) show that the geometry change cannot be solely responsible for the bathochromic shift from 480 to 632 nm of protein-bound astaxanthin. The calculations are consistent with a polarization mechanism that involves the protonation or another interaction with a positive ionic species of comparable magnitude with both ketofunctionalities of the astaxanthin-chromophore and support the changes observed in the resonance Raman and visible absorption spectra. The results are in good agreement with the conclusions that were drawn on the basis of a study of the charge densities in the chromophore in alpha-crustacyanin by solid-state NMR spectroscopy. From the results the dramatic bathochromic shift can be explained not only from a change in the ground electronic state conformation but also from an interaction in the excited electronic state that significantly decreases the energy of the pi-antibonding C==O orbitals and the HOMO-LUMO gap.

Topics: Animals; beta Carotene; Carrier Proteins; Models, Chemical; Models, Molecular; Molecular Structure; Nephropidae; Pigments, Biological; Proteins; Quantum Theory; Spectrum Analysis, Raman; Xanthophylls

Helicobacter pylori is a gram-negative bacterium affecting about half of the world population, causing chronic gastritis type B dominated by activated phagocytes. In some patients the disease evolves into gastric ulcer, duodenal ulcer, gastric cancer or MALT lymphoma. The pathogenesis is in part caused by the immunological response. In mouse models and in human disease, the mucosal immune response is characterized by activated phagocytes. Mucosal T-lymphocytes are producing IFN-gamma thus increasing mucosal inflammation and mucosal damage. A low dietary intake of antioxidants such as carotenoids and vitamin C may be an important factor for acquisition of H. pylori by humans. Dietary antioxidants may also affect both acquisition of the infection and the bacterial load of H. pylori infected mice. Antioxidants, including carotenoids, have anti-inflammatory effects. The aim of the present study was to investigate whether dietary antoxidant induced modulation of H. pylori in mice affected the cytokines produced by H. pylori specific T-cells. We found that treatment of H. pylori infected mice with an algal cell extract containing the antioxidant astaxanthin reduces bacterial load and gastric inflammation. These changes are associated with a shift of the T-lymphocyte response from a predominant Th1-response dominated by IFN-gamma to a Th1/Th2-response with IFN-gamma and IL-4. To our knowledge, a switch from a Th1-response to a mixed Th1/Th2-response during an ongoing infection has not been reported previously.

Topics: Animals; Antioxidants; beta Carotene; Cytokines; Gastritis; Helicobacter Infections; Helicobacter pylori; Interferon-gamma; Interleukin-4; Mice; Mice, Inbred BALB C; Spleen; T-Lymphocytes, Helper-Inducer; Xanthophylls

A 13-week oral repeated dose toxicity study of haematococcus color, a food additive mainly composed of astaxanthin, was conducted in male and female F344 rats. Rats were randomly divided into 4 groups each consisting of 10 males and 10 females and given CRF-1 powder diet containing 0%, 0.025%, 0.075%, and 0.25% haematococcus color, correspond to 0%, 0.5%, 1.5%, and 5% as the product. None of the animals died during the administration period. There were no exposure-related changes in body weight gain or food consumptions. Serum biochemical examinations showed dose-related increase in cholesterol, but the differences were slight and not defined as an adverse effect. No effects related to treatment were noted in hematological examinations and organ weights, and no abnormalities that could be ascribed to exposure to heamatococcus color were observed in histopathological examinations. In conclusion, ingestion of haematococcus color in the diet for 13 weeks does not cause any toxicological changes in F344 rats.

Topics: Animals; beta Carotene; Blood; Body Weight; Eating; Female; Food Coloring Agents; Male; Organ Size; Rats; Rats, Inbred F344; Time Factors; Xanthophylls

An in vitro assay procedure for the carotenoid (beta-ionone ring) 3,3'-hydroxylase and 4,4'-oxygenase has been developed that enables efficient conversion of non-radiolabeled carotenoid substrates added directly into aqueous solution. The following enzymic conversions were demonstrated and apparent kinetic constants (Vmax, Km, and specificity constants) obtained: (a) 3,3'-hydroxylase (from Agrobacterium aurantiacum and Alcaligenes sp. strain PC-1) converted phoenicoxanthin (adonirubin) to astaxanthin, 3-hydroxyechinenone to 4-ketozeaxanthin (adonixanthin), 3'-hydroxyechinenone to 4-ketozeaxanthin, as well as echinenone to 4-ketozeaxanthin via 3- and 3'-hydroxyechinenone; (b) 4,4'-Oxygenase (from A. aurantiacum, Alcaligenes sp. strain PC-1 and Haematococcus pluvialis) converted 4-ketozeaxanthin to astaxanthin, 3-hydroxyechinenone to phoenicoxanthin, 3'-hydroxyechinenone to phoenicoxanthin, and echinenone to canthaxanthin. Determination of substrate specifities allowed assessment of biosynthetic routes to astaxanthin formation and demonstrated that pathways via mono-hydroxylated and ketolated products are enzymically feasible.

Topics: Alcaligenes; Bacteria; beta Carotene; Carotenoids; Chromatography, High Pressure Liquid; Detergents; Kinetics; Lipids; Mixed Function Oxygenases; Molecular Structure; Oxygenases; Rhizobium; Xanthophylls

The food-grade yeast Candida utilis has been engineered to confer a novel biosynthetic pathway for the production of carotenoids such as lycopene, beta-carotene, and astaxanthin. The exogenous carotenoid biosynthesis genes were derived from the epiphytic bacterium Erwinia uredovora and the marine bacterium Agrobacterium aurantiacum. The carotenoid biosynthesis genes were individually modified based on the codon usage of the C. utilis glyceraldehyde 3-phosphate dehydrogenase gene and expressed in C. utilis under the control of the constitutive promotes and terminators derived from C. utilis. The resultant yeast strains accumulated lycopene, beta-carotene, and astaxanthin in the cells at 1.1, 0.4, and 0.4 mg per g (dry weight) of cells, respectively. This was considered to be a result of the carbon flow into ergosterol biosynthesis being partially redirected to the nonendogenous pathway for carotenoid production.

Topics: beta Carotene; Biotechnology; Candida; Carotenoids; Ergosterol; Erwinia; Food Microbiology; Gene Expression; Genes, Bacterial; Lycopene; Plasmids; Rhizobium; Transformation, Genetic; Xanthophylls

The ability of beta-carotene, lutein or astaxanthin to protect against UVA-induced oxidative stress in rat kidney fibroblasts (NRK) was assessed. Activities of the antioxidant enzymes catalase (CAT) and superoxide dismutase (SOD), and changes in thiobarbituric acid reactive substances (TBARS) were measured as indices of oxidative stress. Exposure to UVA light at a dose intensity of 5.6 mW/cm2 for 4 h resulted in a significant decrease in CAT and SOD activities and a significant increase in TBARS. No cytotoxicity, as indicated by lactate dehydrogenase (LDH) release, was observed. beta-Carotene (1 microM), lutein (1 microM) and astaxanthin (10 nM) protect against UVA light-induced oxidative stress in vitro with astaxanthin exhibiting superior protective properties.

Topics: Animals; Antioxidants; beta Carotene; Catalase; Fibroblasts; Kidney; Lutein; Oxidative Stress; Rats; Superoxide Dismutase; Ultraviolet Rays; Xanthophylls

Seven commercial salmon and six trout samples were investigated. Retinol and the carotenoids astaxanthin and canthaxanthin important for pigmentation of the muscle were determined by RP-HPLC. Vitamin A concentrations of raw salmon samples were 16-19 micrograms/100 g, of smoked salmon samples 9-19 micrograms/ 100 g; retinol values of salmon trout (raw) and trout (raw) reached 14-16 micrograms/100 g and 7-9 micrograms/100 g. Concentrations of astaxanthin the important carotenoid of pigmentation, ranged in samples of salmon from 310-465 micrograms/100 g. Samples of salmon trout showed astaxanthin values between 90 and 536 micrograms/100 g, trout samples only 67-85 micrograms/100 g. Concentrations of canthaxanthin were different in the examined samples and were not detectable in all samples. Highest values of canthaxanthin were found in salmon trout samples (113-226 micrograms/100 g), Irish smoked salmon and stremel-salmon (145-169 micrograms/100 g). Raw samples of salmon and of trout showed only low concentrations of canthaxanthin. Astaxanthin and canthaxanthin together reached values of 419-524 micrograms/100 g for salmon, 316-701 micrograms/100 g for salmon trout, and 72-91 micrograms/100 g for trout samples.

Topics: Animals; beta Carotene; Carotenoids; Chromatography, High Pressure Liquid; Fishes; Meat; Meat Products; Salmon; Species Specificity; Trout; Vitamin A; Xanthophylls

Stable red astaxanthin-producing transformants were obtained after genetic transformation of two Phaffia rhodozyma mutants. A yellow mutant, accumulating beta-carotene, and an albino mutant, accumulating phytoene, from P. rhodozyma were transformed using a genomic library of wild-type strain UCD 67-385 in the pBluescript vector. Hybridization assays, using the pBluescript DNA as a radioactive probe, indicate integration of vector sequences into the genome of the transformants. Transformants DNA was digested with restriction endonucleases, ligated with T4 DNA ligase and then used to transform E. coli. Ampicillin resistant plasmids, containing 0.1, 0.2, and 2.5 kb DNA inserts of P. rhodozyma, were rescued from the yeast red transformants. The molecular analysis indicate that transformation has occurred by an integration event of donor DNA into the genome of the host strains.

Topics: Basidiomycota; beta Carotene; Blotting, Southern; Carotenoids; Chromatography, High Pressure Liquid; DNA, Fungal; Mutation; Plasmids; Restriction Mapping; Transformation, Genetic; Xanthophylls

The effect of nicotine and diphenylamine on astaxanthin biosynthesis in Xanthophyllomyces dendrorhous was studied. The effects were analysed under standard and low temperature conditions. It was found that 10 mM-nicotine inhibits the cyclization of lycopene and de novo protein synthesis was not needed to reverse the inhibition. The oxidation of beta-carotene was irreversibly inhibited by 10 microM-diphenylamine while the dehydrogenation of phytoene was reversibly inhibited by 60 microM-diphenylamine. The simultaneous exposure to low temperature (4 degrees C) overcomes the inhibition of beta-carotene oxidation at low diphenylamine concentration.

Topics: Antioxidants; Basidiomycota; beta Carotene; Carotenoids; Cycloheximide; Diphenylamine; Dose-Response Relationship, Drug; Nicotine; Temperature; Time Factors; Xanthophylls

Studies employing time-resolved techniques have shown that beta-carotene, astaxanthin, and lycopene behave quite distinctly with respect to radical quenching and stability, lycopene being the least stable. These results are compatible with the relative effects of the various carotenoids on ultraviolet (UV)-mediated carcinogenesis in mice in which a statistically significant exacerbation by beta-carotene and astaxanthin, but not by lycopene, was observed. Interactions between these carotenoids and vitamin C and E radicals not only provide a chemical basis to explain the failure of beta-carotene to provide benefit in recent clinical trials but suggest that future carotenoid supplementation studies should proceed with caution until carotenoid interactions and radical repair mechanism(s) are elucidated.

Topics: Animals; Anticarcinogenic Agents; beta Carotene; Carcinogenicity Tests; Carotenoids; Dietary Supplements; Female; Free Radicals; Lycopene; Mice; Mice, Hairless; Neoplasms, Radiation-Induced; Random Allocation; Skin Neoplasms; Ultraviolet Rays; Xanthophylls

Isolation and characterization of auxotrophic mutants from wild-type and astaxanthin mutant strains of Phaffia rhodozyma is described. Differences in survival were observed when u.v. irradiation of P. rhodozyma wild-type and astaxanthin mutant strains were incubated in the dark or exposed to photoreactivating light. Ultra-violet mutagenesis was not effective to produce auxotrophic mutants in this yeast. Auxotrophic mutants were obtained with high efficiency through a nystatin enrichment procedure after a N-methyl-N'-nitro-N-nitrosoguanidine (NGT) mutagenic treatment with a 0.12% survivor level. Stringent mutagenetic conditions were needed to obtain P. rhodozyma auxotrophs. The most frequent mutants were ade- and met- in a rather narrow auxotroph spectrum. These results may be associated with a possible diploid condition of this yeast. The high number of adenine auxotrophs obtained in relation to other auxotrophic mutants suggests the possibility of some degree of heterozygosity in the wild-type strain UCD 67-385.

Topics: Anti-Bacterial Agents; Antifungal Agents; beta Carotene; Genetic Complementation Test; Genetic Markers; Light; Mitosporic Fungi; Mutagenesis; Nystatin; Ultraviolet Rays; Xanthophylls; Yeasts

Response surface methodology was applied to optimize the growth of the yeast Phaffia rhodozyma in continuous fermentation using peat hydrolysates as substrate. A second-order, complete, factorial design of the experiments was used to develop empirical models providing a quantitative interpretation of the relationships between the two variables studied, dilution rate and pH. Maximum biomass concentration in the fermentor was obtained by employing the following predicted optimum fermentation conditions: a dilution rate of 0.017/h and a pH level of 7.19. A verification experiment, conducted at previously optimized conditions for maximum biomass volumetric productivity (a dilution rate of 0.022/h, and a pH level of 6.90), produced values for biomass concentration, residual substrate concentration, biomass yield, and biomass volumetric productivity that were very close to the predicted values, indicating the reliability of the empirical model. The concentration of the pigment astaxanthin produced by the yeast under the optimized growth conditions was found to be 544 mg astaxanthin/kg dry cell biomass.

Topics: Basidiomycota; beta Carotene; Biomass; Biotechnology; Fermentation; Models, Biological; Xanthophylls

Penaeus japonicus postlarvae, reared under laboratory conditions, were fed an astaxanthin enriched diet to the investigate carotenoid metabolic capabilities during the postlarval development. Animals fed astaxanthin were found to absorb this carotenoid. The decrease of pigment concentration in the carotenoid starved group is related to the duration of the experimental feeding conditions; the carotenoid depletion depends upon the postlarval status at the starting point. As has been shown for adult prawns, the carotenoid pattern of postlarval stages, regardless of the diet, consists mainly of free and esterified astaxanthin; the relative amounts of these fractions undergo slight variations depending on the diet.

Topics: Adjuvants, Immunologic; Animal Feed; Animals; Aquaculture; beta Carotene; Carotenoids; Energy Metabolism; Larva; Penaeidae; Pigmentation; Xanthophylls

Escherichia coli strains expressing the marine bacteria (Agrobacterium aurantiacum and Alcaligenes sp. strain PC-1) astaxanthin biosynthetic genes (crtZ and W), Haematococcus pluvialis bkt, and Erwinia uredovora crtZ genes were used for in vitro characterization of the respective enzymes. Specific enzyme assays indicated that all of the enzymes are bifunctional, in that the CrtZ enzymes formed zeaxanthin from beta-carotene via beta-cryptoxanthin, as well as astaxanthin from canthaxanthin via phoenicoxanthin (adonirubin). The BKT/CrtW enzymes synthesized canthaxanthin via echinenone from beta-carotene and 4-ketozeaxanthin (adonixanthin) with trace amounts of astaxanthin from zeaxanthin. Comparison of maximum catalytic activities as well as selectivity experiments carried out in the presence of both utilizable substrates indicated that the CrtZ enzymes from marine bacteria converted canthaxanthin to astaxanthin preferentially, whereas the Erwinia CrtZ possessed a favorability to the formation of zeaxanthin from beta-carotene. The CrtW/BKT enzymes were not so defined in their substrate preference, responding readily to fluctuations in substrate levels. Other properties obtained indicated that the enzymes were strictly oxygen-requiring; and a cofactor mixture of 2-oxoglutarate, ascorbic acid, and Fe2+ was beneficial to activity. Based on enzymological data, a predicted pathway for astaxanthin biosynthesis is described, and it is proposed that CrtZ-like enzymes be termed carotenoid 3, (3')-beta-ionone ring hydroxylase and CrtW/BKT carotenoid 4, (4')-beta-ionone ring oxygenase.

Topics: Bacterial Proteins; beta Carotene; Erwinia; Kinetics; Mixed Function Oxygenases; Oxygen; Oxygenases; Rhizobium; Substrate Specificity; Xanthophylls

To determine whether carotenoids can modulate xenobiotic-metabolizing enzymes in mice, catalytic activities of several phase I and phase II enzymes have been measured in liver microsomes and cytosol of male Swiss mice fed diets containing beta-carotene, beta-apo-8'-carotenal, canthaxanthin, or astaxanthin (300 mg/kg diet) or treated with 3-methylcholanthrene (3-MC) (3 times at 50 mg/kg ip) for 15 days. Canthaxanthin increased CYP 1A-dependent activities: ethoxyresorufin O-deethylase (EROD) was increased 3-fold, pentoxyresorufin dealkylase (PROD) was increased 2.5-fold, and methoxyresorufin O-demethylase (MROD) was increased 1.6-fold; these increases were much less than those induced by 3-MC, which induced EROD 49-fold, PROD 10-fold, and MROD 4-fold. 3-MC, but not canthaxanthin, also increased relative liver weight, liver P-450 content, NADH-cytochrome c reductase, and benzoxyresorufin dearylase. The three other carotenoids had little or no effect on phase I enzymes. Among the phase II enzyme activities, only NADPH-quinone reductase was slightly increased by 3-MC and carotenoids, except beta-carotene. Among the three carotenoids that have previously been found to be powerful CYP 1A inducers in the rat, i.e., canthaxanthin, astaxanthin, and beta-apo-8'-carotenal, only canthaxanthin shows some (weak) inducing effect of CYP 1A in the 3-MC-responsive Swiss mice, indicating that the mechanism of CYP 1A induction by carotenoids may not be the same as that by 3-MC. In addition, the fact that beta-carotene has no effect on the tested enzymes does not support the hypothesis that the modulation of xenobiotic metabolism is a possible mechanism for the antimutagenic and anticarcinogenic effects of beta-carotene, which have been demonstrated in several in vivo models in mice.

Topics: Animals; beta Carotene; Canthaxanthin; Carotenoids; Cytochrome P-450 CYP1A1; Cytochrome P-450 CYP2B1; Cytochrome P-450 Enzyme System; Cytosol; Enzyme Induction; Liver; Male; Methylcholanthrene; Mice; Microsomes, Liver; Oxidoreductases; Vitamin A; Xanthophylls; Xenobiotics

The ketocarotenoid astaxanthin is produced by a number of marine bacteria and microalgae. It is synthesized from beta-carotene by the addition of two keto groups to carbons C4 and C4' and two hydroxyl groups to C3 and C3'. The gene, crtO, encoding beta-C-4-oxygenase which converts beta-carotene to canthaxanthin was cloned from the green alga Haematococcus plurialis. We transferred crtO to the cyanobacterium Synechococcus PCC7942, which contains a beta-carotene hydroxylase gene and normally accumulates beta-carotene and zeaxanthin. The genetically engineered cyanobacterium produced astaxanthin as well as other ketocarotenoids. The results confirm that crtO can function in cyanobacteria in conjunction with the intrinsic carotenoid enzymes to produce astaxanthin. Specifically, this finding indicates that beta-carotene hydroxylase, which normally converts beta-carotene to zeaxanthin, can also function in the biosynthesis of astaxanthin. These results provide the first evidence of genetic manipulation of a plant-type carotenoid biosynthesis pathway toward the production of novel carotenoids.

Topics: Bacterial Proteins; beta Carotene; Carotenoids; Chlorophyta; Cloning, Molecular; Cyanobacteria; Gene Transfer Techniques; Genes, Plant; Molecular Sequence Data; Oxygenases; Plasmids; Recombinant Proteins; Restriction Mapping; Xanthophylls

Topics: Antioxidants; Apolipoproteins B; beta Carotene; Copper; Depression, Chemical; Humans; Kinetics; Lipid Peroxidation; Oxidation-Reduction; Xanthophylls

Fluorescence detection of cell membrane potentials is an important technique in neurobiology, cell physiology and pharmaceutical screening, but traditional one-fluorophore indicators either respond too slowly or have limited sensitivity. Recently, we introduced two-component sensors based on the transfer of fluorescence resonance energy from fluorescent lectins bound on one side of the plasma membrane to highly fluorescent oxonol acceptors that electrophorese from one face of the membrane to the other in response to membrane potential.. We have found that fluorescent lectins can often be advantageously replaced in such sensors by fluorescently labeled phospholipids. A coumarinlabeled phosphatidylethanolamine donor and a bis(1,3-dihexyl-2-thiobarbiturate)trimethineoxonol acceptor gave the largest sensitivity of fluorescence ratio (>50% per 100 mV) ever reported. The response was also speeded several-fold by lengthening the mobile dye to the pentamethineoxonol analog, the <0.4 ms time constant of which was shorter than action potential durations. Photodynamic damage due to singlet oxygen was reduced by administering a natural carotenoid, astaxanthin.. Voltage-sensitive fluorescence resonance energy transfer already gives record-setting performance on single cells and will continue to be rationally improvable.

Topics: Animals; beta Carotene; Cell Line; Coumarins; Energy Transfer; Fluorescence; Fluorescent Dyes; Kinetics; Lipid Bilayers; Mammals; Membrane Potentials; Patch-Clamp Techniques; Phosphatidylethanolamines; Photochemistry; Spectrometry, Fluorescence; Thiobarbiturates; Tumor Cells, Cultured; Xanthophylls

Selective isotope enrichment, 13C magic angle spinning (MAS) NMR, and semiempirical quantum chemical modeling, have been used to analyze ligand-protein interactions associated with the bathochromic shift of astaxanthin in alpha-crustacyanin, the blue carotenoprotein complex from the carapace of the lobster Homarus gammarus. Spectra of alpha-crustacyanin were obtained after reconstitution with astaxanthins labeled with 13C at positions 4,4', 12,12', 13,13', or 20,20'. The data reveal substantial downfield shifts of 4.9 and 7.0 ppm at positions 12 and 12' in the complex, respectively. In contrast, at the 13 and 13' positions, small upfield shifts of 1.9 ppm were observed upon binding to the protein. These data are in line with previously obtained results for positions 14,14' (3.9 and 6.8 ppm downfield) and 15,15' (0.6 ppm upfield) and confirm the unequal perturbation of both halves after binding of the chromophore. However, these results also show that the main perturbation is of symmetrical origin, since the chemical shift differences exhibit a similar pattern in both halves of the astaxanthin molecule. A small downfield shift of 2.4 ppm was detected for the 4 and 4' positions. Finally, the 20,20' methyl groups are shifted 0.4 ppm upfield by the protein. The full data set provides convincing evidence that charge polarization is of importance for the bathochromic shift. The NMR shifts are compared with calculated charge densities for astaxanthin subjected to variations in protonation states of the ring-functional groups, as models of ligand-protein interactions. Taking into account the color shift and other available optical data, the current model for the mechanisms of interaction with the protein was refined. The results point toward a mechanism in which the astaxanthin is charged and subject to strong electrostatic polarizations originating from both keto groups, most likely a double protonation.

Topics: Animals; beta Carotene; Carbon Isotopes; Carrier Proteins; Chemical Phenomena; Chemistry, Physical; Magnetic Resonance Spectroscopy; Models, Chemical; Molecular Conformation; Molecular Structure; Nephropidae; Pigments, Biological; Proteins; Xanthophylls

To evaluate the antioxidant effects of beta-carotene and astaxanthin, rat liver microsomes were exposed to a mixture of chelated iron (Fe3+/ADP) and NADPH. The carotenoids (190 pmol/mg protein) were incorporated into some of these microsomal membranes, and phospholipid hydroperoxides (PLOOH), thiobarbituric acid reactive substances (TBARS) and endogenous alpha-tocopherol content were measured over time after the initiation of oxidant stress. In control microsomes, oxidant stress led to accumulation of 1,865 (+/- 371) pmol PLOOH/mg protein during the initial 10-min peroxidation reaction, followed by a more gradual decrease during the subsequent 20-min of reaction. PLOOH accumulation during the initial 10-min reaction period was reduced to 588 (+/- 169) pmol/mg protein with beta-carotene present and 800 (+/- 288) pmol/mg protein with astaxanthin present. During the following 20-min of incubation, PLOOH levels declined in the carotenoid-supplemented microsomes but continued to increase at a slower rate in control preparations. TBARS did not show such large accumulation as observed in PLOOH during the initial 10-min incubation in any microsomal sample. The presence of carotenoids in the microsomal membrane partially inhibited the loss of alpha-tocopherol, especially during the later phase of oxidant stress. When lipid peroxidation is generated by membrane-bound cyt-P450, the specific measurement of PLOOH clearly demonstrates that the presence of carotenoids provides antioxidant protection.

Topics: Animals; beta Carotene; Lipid Peroxidation; Lipid Peroxides; Male; Microsomes, Liver; NADP; Phospholipids; Rats; Rats, Sprague-Dawley; Thiobarbituric Acid Reactive Substances; Vitamin E; Xanthophylls

In this work we establish the chromosomal composition of a wild-type, one astaxanthin and two beta-carotene overproducer strains of the red yeast Phaffia rhodozyma. The method used has been pulsed field gel electrophoresis, which has determined 9 DNA chromosomal bands in the yeast genome. The two largest bands are triplets and two other bands, VI and VIII, seem to be doublets. The size of the chromosomal bands varies between 0.35 and 2.5 Mb, suggesting a genome size of 25 Mb. The technique used, complemented with hybridization assays using specific DNA probes, provides direct information about the genomic organization of P. rhodozyma. We have also cloned and located in chromosomal bands different DNA sequences that code for the translation elongation factor 1 alpha (ef-1 alpha), a 7.6 kb BamHI fragment of repetitive DNA (possibly rDNA) and a randomly chosen fragment (named locus R2). Additionally, we have detected a chromosomal length polymorphism between wild-type strains and mutant strains affecting carotenogenesis obtained in our laboratory.

Topics: Basidiomycota; beta Carotene; Cloning, Molecular; DNA Probes; DNA, Fungal; Electrophoresis, Gel, Pulsed-Field; Genome, Fungal; Karyotyping; Methylnitronitrosoguanidine; Mutagenesis; Nucleic Acid Hybridization; Plasmids; Repetitive Sequences, Nucleic Acid; Xanthophylls

The comparative mechanisms and relative rates of nitrogen dioxide (NO2.), thiyl (RS.) and sulphonyl (RSO2.) radical scavenging by the carotenoid antioxidants lycopene, lutein, zeaxanthin, astaxanthin and canthaxanthin have been determined by pulse radiolysis. All the carotenoids under study react with the NO2. radical via electron transfer to generate the carotenoid radical cation (Car.+). In marked contrast the glutathione and 2-mercaptoethanol thiyl radicals react via a radical addition process to generate carotenoid-thiyl radical adducts [RS-Car].. The RSO2. radical undergoes both radical addition, [RSO2-Car]. and electron abstraction, Car.+. Both carotenoid adduct radicals and radical cations decay bimolecularly. Absolute rate constants for radical scavenging were in the order of approximately 10(7)-10(9) M(-1) s(-1) and follow the sequence HO(CH2)2S. > RSO2. > GS. > NO2.. Although there were some discernible trends in carotenoid reactivity for individual radicals, rate constants varied by no greater than a factor of 2.5. The mechanism and rate of scavenging is strongly dependent on the nature of the oxidising radical species but much less dependent on the carotenoid structure.

Topics: Antioxidants; beta Carotene; Canthaxanthin; Carotenoids; Free Radical Scavengers; Free Radicals; Kinetics; Lutein; Lycopene; Nitrogen Dioxide; Spectrophotometry; Xanthophylls; Zeaxanthins

The effect of oil-extracted astaxanthin from the red crab or langostilla (Pleuroncodes planipes) on the growth and pigmentation of forty five rainbow trouts (Oncorhynchus mykiss) was investigated by feeding a test diet supplemented with 75 mg/kg of astaxanthin during six weeks and compared with a control diet. The oil-extraction process of the pigment is described. Weight, flesh astaxanthin concentration, and color (L*, a*, b*) of the flesh were measured at 0, 3, and 6 weeks. No apparent effect of astaxanthin supplementation was observed on fish development. In spite of the low free astaxanthin amount in the diet (8%), an acceptable carotenoid concentration in the flesh (3.60 +/- 0.78 mg/kg, w/w), and a red hue (H(ab)o = 44.13 +/- 2.36) were obtained at the end of the study. The red hue was strongly correlated with the carotenoid concentration (r = 0.98).

Topics: Animals; beta Carotene; Brachyura; Dietary Supplements; Muscles; Oils; Oncorhynchus mykiss; Pigmentation; Xanthophylls

We report an HPLC method that allows the simultaneous separation of configurational isomers of the predominant cis/trans forms of astaxanthin. The configurational isomers of the all-trans-, and most of the configurational isomers of the 9-cis-, 13-cis- and 15-cis-astaxanthin were separated on a Sumichiral OA-2000 column, which is manufactured and packed in Japan with a Pirkle covalent D-phenylglycine chiral stationary phase (CSP). The large separation of the cis isomers from the all-trans isomers that we report here ensure the suitability of this method for the routine determination of the ratio of the configurational isomers of all-trans-astaxanthin.

Topics: Adjuvants, Immunologic; Animals; beta Carotene; Chromatography, High Pressure Liquid; Glycine; Reproducibility of Results; Salmon; Solvents; Stereoisomerism; Xanthophylls

Growth of the yeast Phaffia rhodozyma was carried out in a simplified medium based on less expensive nutrient sources, such as diluted sugar cane juice, urea, and sodium phosphate. The usual content of the astaxanthin, an oxygenated pink carotenoid useful for fish flesh staining, was improved along with with good cell yields (respective values of > 1300 micrograms/g cells and > 5 g cells/L were observed). Yeast invertase and urease must therefore play an important role in the implementation of low-cost culture media.

Topics: beta Carotene; Carbon; Carotenoids; Cost Control; Culture Media; Molecular Structure; Nitrogen; Xanthophylls; Yeasts

Astaxanthin, a carotenoid without provitamin A activity, enhances murine T-helper (Th) cell clone-mediated antibody (Ab) production with suboptimal antigen (Ag) challenges. It also suppresses interferon-gamma (IFN-gamma) production by cloned murine Th1 cells. beta-Carotene is less effective than astaxanthin. This study evaluates the effects of various carotenoids with various relative polarity, provitamin A activity, and antioxidant activity. Carotenoids tested include astaxanthin, cantaxanthin, zeaxanthin, lutein, and lycopene, and their effects were tested at a concentration at which astaxanthin's effect was most potent. A.E7 and CDC35 cells are used as representative type 1 and type 2 Th cell (Th1 and Th2) clones, respectively. In the Th1 clone, astaxanthin, but not other carotenoids, suppressed IFN-gamma production and increased the number of Ab-secreting cells with the use of primed spleen cells. With cultures of Th1 cells and unprimed spleen cells, astaxanthin and zeaxanthin augmented the number of immunoglobulin M Ab-secreting cells. In the cultures of Th2 clone and primed spleen cells, astaxanthin, but not other carotenoids, enhanced the number of Ab-secreting cells. With unprimed spleen cells, lycopene suppressed Th2 clone-mediated Ab production. Interleukin-5 production by the Th2 clone was not significantly altered with the carotenoids tested, irrespective of the use of unprimed or primed spleen cells. Carotenoid actions on Th cells may vary in each carotenoid and do not seem to be closely associated with carotenoid antioxidant activity or relative polarity.

Topics: Animals; Antibody Formation; beta Carotene; Canthaxanthin; Carotenoids; Clone Cells; Female; Interferon-gamma; Interleukin-5; Lutein; Lycopene; Mice; Mice, Inbred DBA; Spleen; T-Lymphocytes, Helper-Inducer; Xanthophylls; Zeaxanthins

Immunomodulating activities of beta-carotene and carotene-associated carotenoids such as canthaxanthin (beta, beta-carotene-4,4 dione) and astaxanthin (3,3'-dihydroxyl beta, beta-carotene 4,4-dione) were analyzed by in vitro cell culture experiments. (i) beta-Carotene, canthaxanthin and astaxanthin caused significant stimulatory effects on the cell proliferative response of spleen cells and thymocytes from BALB/c mice at the concentrations of 2 x 10(-8) to 10(-7) M, although they showed the activities different from each other. (ii) Astaxanthin exhibited the highest activity on the polyclonal antibody (immunoglobulin M and G) production of murine spleen cells at the concentrations of 2 x 10(-8) to 10(-7) M but beta-carotene did not cause a significant effect at a low concentration (2 x 10(-8) M) although stimulated at a high concentration (2 x 10(-7) M). Canthaxanthin expressed moderate activities at the same concentrations. (iii) All tested carotenoids significantly enhanced the release of interleukin-1 alpha and tumor necrosis factor-alpha from murine peritoneal adherent cells at the concentrations of 2 x 10(-8) to 10(-7) M and the ranks of cytokine-inducing activities were astaxanthin > canthaxanthin > beta-carotene. These results indicate that carotenoids such as beta-carotene, canthaxanthin and astaxanthin have possible immunomodulating activities to enhance the proliferation and functions of murine immunocompetent cells.

Topics: Adjuvants, Immunologic; Animals; Antibody Formation; Antioxidants; B-Lymphocytes; beta Carotene; Canthaxanthin; Carotenoids; Cell Adhesion; Cell Culture Techniques; Cell Division; Interleukin-1; Mice; Mice, Inbred BALB C; Peritoneum; Spleen; T-Lymphocytes; Tumor Necrosis Factor-alpha; Xanthophylls

Astaxanthin, a carotenoid without vitamin A activity, enhances T-dependent antigen (Ag)-specific humoral immune responses. We examined carotenoid actions on T-helper (Th) cell activity in a direct manner with reconstitution experiments; spleen Th cells were replaced with Ag-specific Type 1 and Type 2 (Th1 and Th2) Th cell clones. The Ag for the Th1 and Th2 clones were pigeon cytochrome C and rabbit gamma-globulin, respectively. Astaxanthin and beta-carotene augmented the number of IgM antibody (Ab)-secreting cells when unprimed B cells were incubated with Th clones and stimulated with suboptimal doses of Ag specific for each Th clone. The number of IgG Ab-secreting cells were greater with use of in vivo primed B cells than with unprimed B cells in both Th clones. Astaxanthin but not beta-carotene augmented the number of IgG Ab-secreting cells when primed B cells and Th cell clones were stimulated with suboptimal doses of Ag specific for each Th clone. In the presence of optimal doses of Ag for each Th clone, neither carotenoid augmented the number of Ab-secreting cells. Astaxanthin and beta-carotene may enhance the actions of both Th1 and Th2 cells for humoral immune responses with suboptimal Ag challenges; certain carotenoids may help maintain Ag-mediated immune responses at optimal levels.

Topics: Animals; Antigens; B-Lymphocytes; beta Carotene; Carotenoids; Cells, Cultured; Clone Cells; Columbidae; Cytochrome c Group; Dose-Response Relationship, Drug; Enzyme-Linked Immunosorbent Assay; Female; gamma-Globulins; Immunoglobulin G; Immunoglobulin M; Immunoglobulins; Mice; Mice, Inbred DBA; Rabbits; Spleen; T-Lymphocytes, Helper-Inducer; Vitamin A; Xanthophylls

Topics: beta Carotene; Carotenoids; Industrial Microbiology; Xanthophylls; Yeasts

We succeeded in isolating a novel cDNA involved in astaxanthin biosynthesis from the green alga Haematococcus pluvialis, by an expression cloning method using an Escherichia coli transformant as a host that synthesizes beta-carotene due to the Erwinia uredovora carotenoid biosynthesis genes. The cloned cDNA was shown to encode a novel enzyme, beta-carotene ketolase (beta-carotene oxygenase), which converted beta-carotene to canthaxanthin via echinenone, through chromatographic and spectroscopic analysis of the pigments accumulated in an E. coli transformant. This indicates that the encoded enzyme is responsible for the direct conversion of methylene to keto groups, a mechanism that usually requires two different enzymatic reactions proceeding via a hydroxy intermediate. Northern blot analysis showed that the mRNA was synthesized only in the cyst cells of H. pluvialis. E. coli carrying the H. pluvialis cDNA and the E. uredovora genes required for zeaxanthin biosynthesis was also found to synthesize astaxanthin (3S, 3'S), which was identified after purification by a variety of spectroscopic methods.

Topics: Amino Acid Sequence; Bacterial Proteins; Base Sequence; beta Carotene; Binding Sites; Blotting, Northern; Carotenoids; Chlorophyta; Chromatography, High Pressure Liquid; Circular Dichroism; Conserved Sequence; DNA Mutational Analysis; DNA, Complementary; Escherichia coli; Gene Library; Molecular Sequence Data; Oxygenases; Sequence Analysis, DNA; Sequence Deletion; Sequence Homology, Amino Acid; Xanthophylls

The electrophoretic karyotype of three different strains of Phaffia rhodozyma was determined by contour-clamped homogeneous electric field (CHEF)-gel electrophoresis. Significant differences in electrophoretic karyotyping patterns were found among the three strains studied. Between nine and 17 bands were observed. The size of these bands, based on their migration relative to the chromosomal DNA of Schizosaccharomyces pombe, Hansenula wingei was estimated to be between 0.48 and 3.1 Mb.

Topics: beta Carotene; Carotenoids; Chromosomes, Fungal; Electrophoresis; Karyotyping; Polymorphism, Genetic; Xanthophylls; Yeasts

Solar radiations (UV A and B) can cause epidermis photoaging and skin cancers. These frequently irreversible effects result from the in situ generation of free radicals. However, it has been noted that nutritional factors can modulate photochemical damage, in particular the common carotenoids present in food, which can be considered as potential prophylactic agents against carcinogenesis. We investigated the effect of UV A and B radiations on the skin of the SKH1 hairless mouse fed a diet either lacking in vitamin A or supplemented with retinol, beta-carotene or astaxanthin. The latter is an oxygenated carotenoid (like canthaxanthin) without provitamin A activity and with strong singlet oxygen quenching ability. After analysing of vitamin status of each group (plasma retinol concentrations and hepatic reserves), we searched for UV-induced modifications of polyamine metabolism by measuring epidermal ornithine decarboxylase (ODC) activity and free polyamines concentration (putrescine, spermidine and spermine). In the basal state without irradiation, differences in ODC activity between groups were nonsignificant; but after UV stimulation, ODC increased markedly in the skin of vitamin A-deficient animals, much more than in other groups. Curiously, the addition of astaxanthin or beta-carotene to the regimen containing retinol reduced the protective effect of retinol alone. Regarding polyamines after irradiation, putrescine was significantly increased in the skin of deficient animals, in parallel with ODC activity. However, astaxanthin had a stronger inhibitory effect on putrescine accumulation than retinol, and decreased spermidine and spermine concentrations: this suggests a specific action on transglutaminases.

Topics: Animals; beta Carotene; Carotenoids; Epidermis; Female; Food, Fortified; Liver; Mice; Mice, Hairless; Neoplasms, Radiation-Induced; Nutritional Status; Ornithine Decarboxylase; Polyamines; Putrescine; Random Allocation; Skin; Skin Neoplasms; Spermidine; Spermine; Ultraviolet Rays; Vitamin A; Vitamin A Deficiency; Xanthophylls

A carotenoid biosynthesis gene cluster for the production of astaxanthin was isolated from the marine bacterium Agrobacterium aurantiacum. This cluster contained five carotenogenic genes with the same orientation, which were designated crtW, crtZ, crtY, crtI, and crtB. The stop codons of individual crt genes except for crtB overlapped the start codons of the following crt genes. Escherichia coli transformants carrying the Erwinia uredovora carotenoid biosynthesis genes provide suitable substrates for carotenoid biosynthesis. The functions of the five crt genes of A. aurantiacum were determined through chromatographic and spectroscopic analyses of the pigments accumulated in some E. coli transformants carrying various combinations of the E. uredovora and A. aurantiacum carotenogenic genes. As a result, the astaxanthin biosynthetic pathway is proposed for the first time at the level of the biosynthesis genes. The crtW and crtZ gene products, which mediated the oxygenation reactions from beta-carotene to astaxanthin, were found to have low substrate specificity. This allowed the production of many presumed intermediates of astaxanthin, i.e., adonixanthin, phoenicoxanthin (adonirubin), canthaxanthin, 3'-hydroxyechinenone, and 3-hydroxyechinenone.

Topics: Amino Acid Sequence; Base Sequence; beta Carotene; Carotenoids; Codon; Erwinia; Escherichia coli; Genes, Bacterial; Models, Chemical; Molecular Sequence Data; Multigene Family; Restriction Mapping; Rhizobium; Sequence Analysis, DNA; Substrate Specificity; Xanthophylls

The yeast Phaffia rhodozyma, a promising microbial producer of the carotenoid astaxanthin, was cultivated in batch and continuous processes in an agitated and aerated fermenter using an acid peat extract based culture medium. For the accelerated growth phase, the mean specific growth rate and doubling time were found to be 0.038 h-1, and 18.24 hours, respectively. The production of astaxanthin was found to be basically growth associated, the maximum concentrations of the pigment produced in batch culture and continuous cultivation being similar.

Topics: beta Carotene; Biomass; Carotenoids; Fermentation; Kinetics; Soil; Xanthophylls; Yeasts

Carotenoids have recently received considerable interest because of their potential in delaying or preventing degenerative diseases such as arteriosclerosis, cancer, and aging. In this study we show that the active oxygen species singlet oxygen (1O2) and peroxyl radicals differently affect carotenoid composition and biosynthesis in the yeast Phaffia rhodozyma. Photochemical generation of 1O2 with rose bengal or alpha-terthienyl induced carotenoid accumulation. In contrast, peroxyl radicals derived from t-butylhydroperoxide (tBOOH) or H2O2 decreased the content of astaxanthin and increased beta-carotene by approximately 4-fold, suggesting end product feedback regulation by astaxanthin or inhibition of biosynthetic enzymes. 14C labeling of carotenoids during oxidative stress supported the possibility of end product regulation. Carotenoids were bleached by 8 mM tBOOH within 6 h when carotenogenesis was inhibited by thymol. When treated with peroxides, a previously unreported pigment in P. rhodozyma was formed. The carotenoid had a mass of 580 Da and a molecular formula of C40H52O3. Chemical derivatizations combined with mass and absorbance spectroscopy tentatively identified the carotenoid as dehydroflexixanthin (3,1'-dihydroxy-2,3,3',4'-tetradehydro-1',2'-dihydro-beta,psi-caro tene-4-one). This study provides the first report of induction of astaxanthin biosynthesis by 1O2, probable feedback control by astaxanthin, and the oxidative degradation of astaxanthin to novel pigments in P. rhodozyma.

Topics: beta Carotene; Carotenoids; Free Radicals; Mevalonic Acid; Oxygen; Peroxides; Photochemistry; Reactive Oxygen Species; Singlet Oxygen; tert-Butylhydroperoxide; Xanthophylls; Yeasts

The effect of carotenoids on in vitro immunoglobulin (Ig) production by peripheral blood mononuclear cells (PBMNC) was examined by employing blood samples from adult volunteers and full-term newborn babies (umbilical cord blood). Under carotenoid-supplemented culture conditions, cells were stimulated by polyclonal stimulants, neoantigens, and a recall antigen (Ag), and IgM, IgA, and IgG levels in the culture supernatant were measured. Beta-carotene and astaxanthin were used as representatives of carotenoids with and without vitamin A activity, respectively. Astaxanthin enhanced IgM production in response to T-dependent Ag (TD-Ag) and a T-dependent polyclonal stimulant. Astaxanthin also augmented IgG production in response to a recall Ag. IgA production without supplemental carotenoids was negligible for all stimuli. However, in carotenoid-supplemented cultures, IgA production was significantly higher in response to a T-dependent polyclonal stimulant than in unsupplemented cultures. IgM and IgA production was augmented at 10(-8) mol/l astaxanthin, whereas astaxanthin enhanced IgG production in response to a recall Ag at 10(-10)-10(-9) mol/l. Similar enhancing actions of astaxanthin on IgM production were observed in cord blood mononuclear cells (CBMNC), although CBMNC produced less IgM than adult PBMNC. Beta-carotene did not have a significant effect on human Ig production. The carotenoid actions were not demonstrated under serum-free culture conditions; serum is essential for solubilization of carotenoids. In summary, this study has shown for the first time that astaxanthin, a carotenoid without vitamin A activity, enhances human Ig production in response to T-dependent stimuli.

Topics: Adult; Antibody Formation; beta Carotene; Blood; Carotenoids; Cells, Cultured; Dose-Response Relationship, Drug; Female; Fetal Blood; Humans; Immunoglobulin A; Immunoglobulin G; Immunoglobulin M; Infant, Newborn; Leukocytes, Mononuclear; Male; T-Lymphocytes; Xanthophylls

The chemopreventive effects of two xanthophylls, astaxanthin (AX) and canthaxanthin (CX) on oral carcinogenesis induced by 4-nitroquinoline 1-oxide (4-NQO) was investigated in male F344 rats. Rats were given 20 ppm of 4-NQO in their drinking water for 8 weeks to induce oral neoplasms or preneoplasms. Animals were fed diets containing 100 ppm AX or CX during the initiation or postinitiation phase of 4-NQO-induced oral carcinogenesis. The others contained the groups of rats treated with AX or CX alone and untreated. At the end of the study (week 32), the incidences of preneoplastic lesions and neoplasms in the oral cavity of rats treated with 4-NQO and AX or CX were significantly smaller than those of rats given 4-NQO alone (P < 0.001). In particular, no oral neoplasms developed in rats fed AX and CX during the 4-NQO exposure and in those given CX after the 4-NQO administration. Similarly, the incidences of oral preneoplastic lesions (hyperplasia and dysplasia) in rats treated with 4-NQO and AX or CX were significantly smaller than that of the 4-NQO-alone group (P < 0.05). In addition to such tumor inhibitory potential, dietary exposure of AX or CX decreased cell proliferation activity in the nonlesional squamous epithelium exposed to 4-NQO as revealed by measuring the silver-stained nucleolar organizer regions protein number/nucleus and 5'-bromodeoxyuridine-labeling index. Also, dietary AX and CX could reduce polyamine levels of oral mucosal tissues exposed to 4-NQO. These results indicate that AX and CX are possible chemopreventers for oral carcinogenesis, and such effects may be partly due to suppression of cell proliferation.

Topics: Animals; Anticarcinogenic Agents; beta Carotene; Bromodeoxyuridine; Canthaxanthin; Carotenoids; Male; Mouth Neoplasms; Nucleolus Organizer Region; Precancerous Conditions; Rats; Rats, Inbred F344; Xanthophylls

MAS (magic angle spinning) 13C NMR has been used to study protein-chromophore interactions in alpha-crustacyanin, the blue astaxanthin-binding carotenoprotein of the lobster, Homarus gammarus, reconstituted with astaxanthins labelled with 13C at the 14,14' or 15,15' positions. Two signals are seen for alpha-crustacyanin containing [14,14'-13C2]astaxanthin, shifted 6.9 and 4.0 ppm downfield from the 134.1 ppm signal of uncomplexed astaxanthin in the solid state. With alpha-crustacyanin containing [15,15'-13C2]astaxanthin, one essentially unshifted broad signal is seen. Hence binding to the protein causes a decrease in electronic charge density, providing the first experimental evidence that a charge redistribution mechanism contributes to the bathochromic shift of the astaxanthin in alpha-crustacyanin, in agreement with inferences based on resonance Raman data [Salares, et al. (1979) Biochim. Biophys. Acta 576, 176-191]. The splitting of the 14 and 14' signals provides evidence for asymmetric binding of each astaxanthin molecule by the protein.

Topics: Animals; beta Carotene; Carotenoids; Carrier Proteins; Magnetic Resonance Spectroscopy; Nephropidae; Pigments, Biological; Protein Conformation; Proteins; Xanthophylls

The modulating effects of dietary feeding of two xanthophylls, astaxanthin (AX) and canthaxanthin (CX) during the postinitiation phase on colon carcinogenesis initiated with azoxymethane (AOM) were investigated in male F344 rats. Animals were initiated with AOM by weekly s.c. injections of 15 mg/kg body wt for 3 weeks and then they were fed the diets containing AX or CX at concentrations of 100 and 500 p.p.m. for 34 weeks. The others contained the groups of rats treated with AX or CX alone and untreated. At the end of the study (week 37), the incidence and multiplicity of neoplasms (adenoma and adenocarcinoma) in the large intestine of rats initiated with AOM and followed by AX or CX containing diet at a high dose (500 p.p.m.) were significantly smaller than those of rats given AOM alone (P < 0.001). In addition, AX or CX feeding significantly inhibited the development of aberrant crypt foci induced by AOM. Dietary exposure to AX or CX also decreased cell proliferation activity as revealed by measuring 5'-bromodeoxyuridine-labeling index as crypt cells, colonic mucosal ornithine decarboxylase activity and blood polyamine levels. These results indicate that AX and CX are possible chemopreventers for carcinogenesis of colon in addition to urinary bladder and oral cavity and such effects may be partly due to suppression of cell proliferation.

Topics: Animals; Azoxymethane; beta Carotene; Bromodeoxyuridine; Canthaxanthin; Carcinogens; Carotenoids; Colon; Colonic Neoplasms; Diet; Incidence; Intestinal Mucosa; Male; Ornithine Decarboxylase; Polyamines; Rats; Rats, Inbred F344; Xanthophylls

A physical chemistry technique based on singlet oxygen luminescence at about 1270 nm and a biological cell membrane technique were used to study the quenching of singlet oxygen by four carotenoids bound to the surface of lymphoid cells. All the carotenoids studied showed a beneficial effect in cell protection, but there were subtle differences between them.

Topics: Administration, Oral; beta Carotene; Canthaxanthin; Carotenoids; Cell Survival; Cells, Cultured; Diet; Humans; Kinetics; Luminescent Measurements; Lycopene; Lymphocytes; Oxygen; Singlet Oxygen; Time Factors; Xanthophylls

Carotenoids stabilize beta-carotene 15,15'-dioxygenase (CDO) during the isolation of the enzyme from the rabbit small intestinal mucosa using a thiol protector and protease inhibitors and in the course of the enzyme purification. The inhibition of CDO by lycopene, lutein and astaxanthin is competitive. Carotenoids partly protect CDO from the action of both iodacetate and trypsin due to enzyme-pseudosubstrate interaction.

Topics: Animals; beta Carotene; beta-Carotene 15,15'-Monooxygenase; Binding, Competitive; Carotenoids; Dithiothreitol; Enzyme Stability; Intestinal Mucosa; Iodoacetates; Iodoacetic Acid; Lutein; Lycopene; Oxygenases; Rabbits; Trypsin; Xanthophylls

Previously, we demonstrated an enhancement of in vitro antibody (Ab) production in response to T-dependent antigens (TD-Ag) by astaxanthin, a carotenoid without vitamin A activity. The effects of beta-carotene, a carotenoid with vitamin A activity, and lutein, another carotenoid without vitamin A activity, on in vitro Ab production were examined with spleen cells from young and old B6 mice. In addition, the in vivo effects of lutein, astaxanthin, and beta-carotene on Ab production were studied in young and old B6 mice. Lutein, but not beta-carotene, enhanced in vitro Ab production in response to TD-Ags. The depletion of T-helper cells prevented the enhancement of Ab production by lutein and astaxanthin. In vivo Ab production in response to TD-Ag was significantly enhanced by lutein, astaxanthin, and beta-carotene. The numbers of immunoglobulin M- and G-secreting cells also increased in vivo with the administration of these carotenoids when mice were primed with TD-Ags. Antibody production in response to TD-Ags in vivo and in vitro was significantly lower in old than in young B6 mice. Astaxanthin supplements partially restored decreased in vivo Ab production in response to TD-Ags in old B6 mice. Lutein and beta-carotene also enhanced in vivo Ab production in response to TD-Ags in old B6 mice, although to a lesser extent than did astaxanthin. However, none of the carotenoids had an effect on in vivo or in vitro Ab production in response to T-independent antigen. These results indicate significant immunomodulating actions of carotenoids for humoral immune responses to TD-Ags and suggest that carotenoid supplementation may be beneficial in restoring humoral immune responses in older animals.

Topics: Adjuvants, Immunologic; Age Factors; Animals; Antibody Formation; Antigens; Antigens, T-Independent; beta Carotene; Carotenoids; Dose-Response Relationship, Drug; Enzyme-Linked Immunosorbent Assay; Female; Immunoglobulin G; Immunoglobulin M; In Vitro Techniques; Mice; Mice, Inbred C57BL; T-Lymphocytes; T-Lymphocytes, Helper-Inducer; Xanthophylls

The chemopreventive effects of two xanthophylls, astaxanthin (AX) and canthaxanthin (CX), on urinary bladder carcinogenesis induced by N-butyl-N(4-hydroxybutyl)nitrosamine (OH-BBN) was investigated in male ICR mice. Mice were given 250 p.p.m. OH-BBN in drinking water for 20 weeks and after a 1 week interval with tap water, water containing AX or CX at a concentration of 50 p.p.m. was administered during subsequent 20 weeks. Other groups of mice were treated with AX or CX alone or untreated. At the end of the study (week 41), the incidences of preneoplastic lesions and neoplasms in the bladder of mice treated with OH-BBN and AX or CX were smaller than those of mice given OH-BBN. In particular, AX administration after OH-BBN exposure significantly reduced the incidence of bladder cancer (transitional cell carcinoma) (P < 0.003). However, the inhibition of the frequencies of such lesions in mice treated with OH-BBN and CX was not significant. Treatment with AX or CX also decreased the number/nucleus of silver-stained nucleolar organizer region proteins (AgNORs), a new index of cell proliferation, in the transitional epithelium exposed to OH-BBN. Preneoplasms and neoplasms induced by OH-BBN, and the antiproliferative potential, was greater for AX than CX. These results indicate that AX is a possible chemopreventive agent for bladder carcinogenesis and such an effect of AX may be partly due to suppression of cell proliferation.

Topics: Animals; Anticarcinogenic Agents; beta Carotene; Body Weight; Butylhydroxybutylnitrosamine; Canthaxanthin; Carotenoids; Liver; Male; Mice; Mice, Inbred ICR; Nucleolus Organizer Region; Organ Size; Precancerous Conditions; Silver Staining; Urinary Bladder Neoplasms; Xanthophylls

Farming of trout requires a finishing diet containing pigments such as astaxanthin and canthaxanthin so that they may achieve a pink tissue coloration similar to that of wild trout. The production of synthetic pigments is not enough so that the requirements of the aquaculture industry are not met, besides having a high cost. Thus, the objective of the present study was to evaluate the deposition of saponified and esterified chili (Capsicum annuum) extracts in the skin and muscle of rainbow trout (Oncorhynchus mykiis). The experiment consisted of three treatments with the inclusion in the finishing diet of saponified and esterified chili extracts and of astaxanthin, respectively. Each treatment was carried out with 150 organisms with an average weight of a 150 g and done in duplicate. The results showed that both the saponified and esterified chili extracts pigmented the skin and muscle of rainbow trout, although the pigmentation effect was less red than that produced by the astaxanthin control.

Topics: Animal Feed; Animals; Aquaculture; beta Carotene; Capsicum; Carotenoids; Esterification; Muscle, Skeletal; Oncorhynchus mykiss; Pigmentation; Plant Extracts; Plants, Medicinal; Skin; Skin Pigmentation; Weight Gain; Xanthophylls

Large unilamellar liposomes comprising of egg yolk phosphatidylcholine (PC) was exposed to photoirradiation in the presence of methylene blue (water-soluble photosensitizer) or 12-(1-pyrene)dodecanoic acid (P-12, lipid-soluble photosensitizer) to estimate the inhibitory effect of beta-carotene and astaxanthin on photosensitized oxidation of phospholipid bilayers. Without sensitizers, astaxanthin decreased much slower than beta-carotene and other hydrocarbon carotenoids (lycopene, alpha-carotene). Astaxanthin lasted longer than beta-carotene even in the presence of methylene blue or P-12. Decrease of astaxanthin was also much slower than that of beta-carotene when egg yolk PC was replaced by dimyristoyl PC. However, inhibitory effect of astaxanthin was lower than beta-carotene in the case of P-12 sensitized photooxidation. These results suggest that effectiveness of carotenoids as antioxidants on photosensitized oxidation (Type II) in phospholipid bilayers depends on the site of singlet oxygen to be generated, as well as their stability on photoirradiation.

Topics: beta Carotene; Carotenoids; Lauric Acids; Lipid Bilayers; Lipid Peroxidation; Liposomes; Methylene Blue; Oxidation-Reduction; Phosphatidylcholines; Photosensitizing Agents; Vitamin E; Xanthophylls

The chemopreventive action of carotenoids on proteinuria and lymphadenopathy were examined in autoimmune-prone MRL-lpr/lpr (MRL/l) mice. They were fed a synthetic full-fed diet (16-18 kcal/mouse/day) with supplementation of beta-carotene or astaxanthin (0.19 mumoles/mouse, 3 times a week), and the development of lymphadenopathy and proteinuria were examined. MRL/l mice fed a full-fed diet without the supplementation of carotenoids or those fed a calorie-restricted (CR) diet (10-11 kcal/mouse/day, 60% calorie intake of full-fed mice) were employed as controls. CR dramatically delayed the development of proteinuria and lymphadenopathy, as reported previously. Carotenoids also significantly delayed the onset of these symptoms in MRL/l mice fed a full-fed diet. Carotenoids were half as effective as CR and astaxanthin, a carotenoid without provitamin A activity, which appeared to exert more significant preventive actions than beta-carotene in delaying the development of these symptoms. Similar chemopreventive actions of carotenoids were also demonstrated in MRL/l mice fed a regular diet (Lab Chow). CR has been shown to augment IL-2 production and to decrease serum prolactin levels in this strain, which may be related to its dramatic preventive action of autoimmunity. However, carotenoids did not affect IL-2 production nor prolactin levels in full-fed MRL/l mice. The chemopreventive actions of carotenoids observed in autoimmune-prone MRL/l mice may be attributed to yet unknown mechanisms, apart from their provitamin A activity or oxygen-quenching activity.

Topics: Animals; Autoimmune Diseases; beta Carotene; Body Weight; Carotenoids; Diet, Reducing; Energy Intake; Female; Food, Fortified; Interleukin-2; Lymph Nodes; Lymphocyte Activation; Lymphoproliferative Disorders; Mice; Mice, Mutant Strains; Organ Size; Prolactin; Proteinuria; T-Lymphocytes; Xanthophylls

The lipid and fatty acid composition of the hepatopancreas and muscle of the prawn, Penaeus japonicus, were analyzed. The hepatopancreas was the main lipid storage organ, triglycerides and phospholipids being its major lipid components, while muscle contained mainly phospholipids. The fatty acid compositions of total lipids from the hepatopancreas and muscle were similar to those in other marine animals. The major fatty acids were palmitic (16:0), oleic (18:1n-9), eicosapentaenoic (20:5n-3), and docosahexaenoic (22:6n-3) acids. The monoglycerides, diglycerides, triglycerides, and cholesterol esters from the hepatopancreas and muscle exhibited similar fatty acid patterns, but each lipid fraction was characterized by a specific paraffin chain composition. A blue carotenoprotein (lambda max = 640 nm) containing astaxanthin was also extracted and purified from the hypodermis of the prawn. This blue carotenoprotein has a molecular weight of ca. 280,000, which is much lower to those given for other crustaceans. The carotenoid prosthetic group was released from the carotenoprotein by the addition of acetone, and showed a hypsochromic shift to 470 nm and the characteristic shape of free ketocarotenoids. TLC, infrared spectroscopy, chemical reduction, spectrophotometry, and qualitative analysis by HPLC were used to identify the astaxanthin as a unique chromophore group of the blue carotenoprotein. Moreover, HPLC studies suggested all-trans-astaxanthin to be the main component, which was accompanied by an epimer and its cis-isomer.

Topics: Animals; beta Carotene; Carotenoids; Chromatography, High Pressure Liquid; Eicosapentaenoic Acid; Fatty Acids; Lipids; Liver; Molecular Weight; Muscles; Oleic Acid; Oleic Acids; Palmitic Acid; Palmitic Acids; Pancreas; Penaeidae; Proteins; Xanthophylls

Interaction of rabbit small intestine mucosa beta-carotene 15,15'-dioxygenase with lycopene, 15,15'-dehydro-beta-carotene, lutein and astaxanthine is unaccompanied by the formation of products of enzymatic cleavage of the central double bond of these carotenoid molecules. Apo-carotenoids of lutein and lycopene are formed by nonenzymatic oxidation. Lycopene and 15,15'-dehydro-beta-carotene competitively inhibit the enzymatic oxidation of beta-carotene. Inhibition of the enzyme conversion by lutein and astaxanthin is less strong, being of a competitive-noncompetitive type. Enzymatic formation of retinal from beta-carotene depends on the accessibility of substrate molecules.

Topics: Animals; beta Carotene; beta-Carotene 15,15'-Monooxygenase; Carotenoids; Intestinal Mucosa; Intestine, Small; Lutein; Lycopene; Oxygenases; Rabbits; Retinaldehyde; Substrate Specificity; Xanthophylls

Previously we have shown that astaxanthin, a carotenoid without provitamin A activity, enhances in vitro antibody (Ab) production to sheep red blood cells in normal B6 mice. In this study, we further attempted to examine the mechanisms of this enhancing action of carotenoids on specific Ab production in vitro in relation to different antigen (Ag) stimuli, cytokine production, and T- and B-cell interactions in both normal and autoimmune strains of mice. When the actions of carotenoids were tested in normal strains of mice, we found that astaxanthin enhanced in vitro Ab production to T cell-dependent Ag, but not to T-independent Ag, and did not augment total immunoglobulin production. Astaxanthin exerted maximum enhancing actions when it was present at the initial period of Ag priming. This action of astaxanthin was abolished when T cells were depleted from spleen cell suspensions and appeared to require direct interactions between T and B cells. The results also indicated that carotenoids may modulate the production of interferon-tau in this assay system. When the actions of carotenoids were tested in autoimmune-prone MRL and NZB mice, the enhancing action of astaxanthin on in vitro Ab production was less significant. Furthermore, carotenoids did not potentiate or augment spontaneous Ab and immunoglobulin production by spleen cells in these strains. Taken together, carotenoids without provitamin A activity may be able to augment in vitro specific Ab production to T cell-dependent Ag partly through affecting the initial stage of Ag presentation without facilitating polyclonal B-cell activation or autoantibody production.

Topics: Adjuvants, Immunologic; Animals; Antibody Formation; Autoimmune Diseases; B-Lymphocytes; beta Carotene; Carotenoids; Cells, Cultured; Cytokines; Lymphocyte Activation; Mice; Mice, Inbred Strains; T-Lymphocytes; Xanthophylls

Carotenoid scavenging of free radicals has been investigated in peroxidizing methyl esters of unsaturated fatty acids using (i) metmyoglobin as a water-based free-radical initiator in a heterogeneous lipid/water system, and (ii) azo-bis-isobutyronitrile as a free-radical initiator in a homogeneous chloroform solution. For the heterogeneous system, using a combination of electrochemical oxygen depletion measurements, spectrophotometric determination of lipid hydroperoxides and carotenoid degradation, it was demonstrated that each of the four carotenoids astaxanthin, beta-carotene, canthaxanthin, and zeaxanthin protects the methyl esters against oxidation. The antioxidative effect increases with increasing carotenoid concentration, increases with decreasing oxygen partial pressure (0.010 < pO2 < 0.50 atm), and shows little dependence on the structure of the carotenoid. For a homogeneous solution, the effect of the structure of the carotenoid was further investigated, and it was shown that the stability of the four carotenoids in the oxidizing system are different, with the order of decreasing stability being: astaxanthin > canthaxanthin > beta-carotene > zeaxanthin. Each of the four carotenoids can suppress lipid oxidation and the degree of suppression of peroxidation of methyl linoleate corresponds to the difference in stability.

Topics: Adipose Tissue; beta Carotene; Canthaxanthin; Carotenoids; Free Radical Scavengers; Kinetics; Linoleic Acids; Linolenic Acids; Lipid Metabolism; Metmyoglobin; Models, Biological; Nitriles; Oxidation-Reduction; Oxygen; Oxygen Consumption; Structure-Activity Relationship; Xanthophylls; Zeaxanthins

Three groups of male Wistar rats (130-140 g) were fed 30 days with a synthetic diets containing 0.1% of beta-carotene, canthaxanthin and astaxanthin respectively. Another group was fed with a synthetic diet without carotenoids. The results shows that the beta-carotene does not induce change in plasma cholesterol (49, 7 +/- 3.6 mg/dl), but canthaxanthin and astaxanthin induce a significant increase in cholesterol concentration (92.1 +/- 3.6 and 66.5 +/- 5.1 mg/dl). This increase is noted mainly in the HDL fraction of the lipoproteins. Canthaxanthin has more affinity than astaxanthin for the liver, principal site of lipoproteins catabolism. The hypercholesterolemic effect of these xanthophylls is not related to reported mechanisms of carotenoids in mammalian, because beta-carotene does not induce changes in plasma cholesterol.

Topics: Animals; beta Carotene; Canthaxanthin; Carotenoids; Hypercholesterolemia; Liver; Male; Rats; Rats, Wistar; Tissue Distribution; Xanthophylls

Phaffia rodozyma CCRC-21346, CBS-6938, and CBS-5908 were treated with mutagenic agent NTG (N-methyl-N'-nitro-N-nitrosoguanidine) several times, then they were plated onto yeast-malt agar containing beta-ionone as a selective medium. Several isolates had increased astaxanthin content compared with the parental natural isolates. NCHU-FS301, one of the NTG treated strains, produced considerably more astaxanthin (1515.63 micrograms/g yeast) than the parent CBS-6938 (565.08 micrograms/g yeast). In studying the effects of carbon sources on the red pigment formation, it was found that glucose supported the highest total astaxanthin production (7809.3 micrograms/l). Yeast extract was the best nitrogen source in supporting the highest total astaxanthin formation (8637.5 micrograms/l). Beef extract, yeast extract, and potassium nitrate added in an equal ratio as a nitrogen source supported more pigment formation (8052.6 micrograms/l) than the rest of the mixture tested. Using kinetic parameters of specific growth rate (mu) and specific astaxanthin productivity (qp) to judge the association between growth behavior and product formation, the NCHU-FS301 showed more positive growth-associated fermentation type than the parent strain. These astaxanthin-overproducing mutants could be useful for the aquacultural industry in providing a natural source of astaxanthin.

Topics: beta Carotene; Carbon; Carotenoids; Culture Media; Fermentation; Hydrogen-Ion Concentration; Kinetics; Mutagenesis; Nitrogen; Temperature; Xanthophylls; Yeasts

When the conjugated keto-carotenoids, either astaxanthin or canthaxanthin, are added to rat liver microsomes undergoing radical-initiated lipid peroxidation under air, they are as effective as alpha-tocopherol in inhibiting this process. This contrasts with the effect of beta-carotene, which is a much less potent antioxidant when added in this system, without the addition of other antioxidants.

Topics: Adjuvants, Immunologic; Animals; Antioxidants; beta Carotene; Canthaxanthin; Carotenoids; Kinetics; Lipid Peroxidation; Malondialdehyde; Microsomes, Liver; NADP; Rats; Rats, Inbred Strains; Vitamin E; Xanthophylls

1. One force-fed meal containing labelled 14C-astaxanthin (14C-Ax) and 3H-canthaxanthin (3H-Cx) or 3H-zeaxanthin (3H-Zx) was given to eight mature female rainbow trout. Ninety-six hours after the test meal ingestion, trout were killed and liver, skin, muscle and ovaries were dissected out. 2. Ax accumulated slightly more in muscle than Cx but in all tissues Ax and Cx were very significantly more concentrated than Zx. 3. 3H-Zx metabolites were found only in the liver, whereas 14C-phoenicoxanthin was the only metabolic pigment from 14C-Ax detected and was found in all investigated tissues. 4. 3H-Ax was found in the liver of all trout indicating that 3H-Cx and 3H-Zx were Ax precursors, and that salmonids probably possess carotenoid oxidative pathways unknown until now. 5. Labelled retinol1 and retinol2 were detected only in the liver and 3H-Zx was largely the predominant precursor of these two vitamin A forms.

Topics: Animals; beta Carotene; Biotransformation; Canthaxanthin; Carotenoids; Diet; Female; Tissue Distribution; Trout; Vitamin A; Xanthophylls; Zeaxanthins

The carotenoid pigment astaxanthin (3,3'-dihydroxy-beta,beta-carotene-4,4'-dione) is an important component in feeds of aquacultural animals. It is produced as a secondary metabolite by the yeast Phaffia rhodozyma, and the isolation of rare mutants that produce increased quantities is limited by the lack of genetic selections. As a model system for enriching mutants increased in production of secondary metabolites, we have used quantitative flow cytometry/cell sorting (FCCS) to isolate astaxanthin hyperproducing mutants of the yeast. Experimental conditions were developed that gave a quantitative correlation of fluorescence and carotenoid content. In mutated populations, a 10,000-fold enrichment of carotenoid-overproducing yeasts was obtained. Distinctive differences were detected by FCCS in fluorescence and forward scatter values of mutant and wild-type populations of yeasts. Comparison of wild-type and mutant clones by fluorescence confocal laser microscopy showed that the mutants had more intense fluorescence throughout the cell than the wild-type. Quantitative FCCS is a sensitive method to isolate and characterize carotenoid overproducing mutants and should be useful as a general method for the isolation of mutants increased in other fluorescent metabolites.

Topics: beta Carotene; Carotenoids; Cell Separation; Flow Cytometry; Fluorescence; Microscopy, Fluorescence; Mutation; Xanthophylls; Yeasts

1. Bovine serum albumin (BSA) and/or egg albumin were bound to astaxanthin or canthaxanthin easily and the spectroscopic characteristics of these complexes were similar to those of astaxanthin or canthaxanthin in the salmon muscle. 2. This result indicates that astaxanthin-BSA, -egg albumin, canthaxanthin-BSA and -egg albumin complexes were basically similar to astaxanthin-actomyosin and/or canthaxanthin-actomyosin complex in the salmon muscle. 3. The binding of salmon actomyosin to astaxanthin or canthaxanthin is not specific.

Topics: Actomyosin; Animals; beta Carotene; Canthaxanthin; Carotenoids; Circular Dichroism; Muscles; Ovalbumin; Oxidation-Reduction; Salmon; Serum Albumin, Bovine; Spectrum Analysis, Raman; Xanthophylls

The immunomodulating effects of carotenoids (beta-carotene and astaxanthin) on mouse lymphocytes were studied in in vitro culture system by use of assay for mitogen responses of spleen cells, thymocyte proliferation, interleukin 2 production, and antibody (Ab) production in vitro in response to sheep red blood cells. Changes of cell surface markers on spleen lymphocytes including Ia antigen (Ag), surface immunoglobulin, B220, and Thy-1 Ag were also examined. At a concentration of 10(-8) M, carotenoids did not show any significant effect on mitogen responses (phytohemagglutinin P and concanavalin A) on murine spleen cells, irrespective of the concentrations of mitogens used. Interleukin 2 production by murine spleen cells was not significantly altered by carotenoids in the culture media (10(-7) to 10(-9) M). [3H]thymidine incorporation by B6 thymocytes was somewhat enhanced in the presence of astaxanthin or beta-carotene when cultured in the concentration of 10(6)/ml. At higher concentrations of cells (5 x 10(6)/ml), such an effect was not observed. In assays of in vitro Ab production in response to sheep red blood cells, B6 spleen cells produced significantly more Ab-forming cells (plaque-forming cells, immunoglobulins M and G) in the presence of astaxanthin (greater than 10(-8) M) but not beta-carotene. Expression of Ia Ag seemed to be moderately enhanced on both Thy-1+ and Thy-1- spleen cells in the presence of astaxanthin (greater than 10(-9) M) but not beta-carotene. The expression of Thy-1 and surface immunoglobulin seemed unchanged with the treatment of these carotenoids. These results indicate that immunomodulating actions of carotenoids are not necessarily related to provitamin A activity, because astaxanthin, which does not have provitamin A activity, showed more significant effects in these bioassays and also indicate that such actions of carotenoid demonstrated in this study may be difficult to explain only by its oxygen-quenching capacity.

Topics: Adjuvants, Immunologic; Animals; Antibody Formation; Antigens, Surface; beta Carotene; Carotenoids; Cell Division; Cells, Cultured; Erythrocytes; Female; Interleukin-2; Lymphocytes; Mice; Mice, Inbred Strains; Mitogens; Thymus Gland; Xanthophylls

1. The main carotenoids in wild Penaeus monodon exoskeleton were astaxanthin di- and mono-esters, astaxanthin, and beta-carotene. 2. Wild P. monodon exoskeleton contained on average 26.3 ppm total carotenoid; normally pigmented farmed shrimp had a similar concentration (25.3 ppm). 3. Exoskeletons of farmed "blue" P. monodon (i.e. blue-coloured, as opposed to the normally red-blue/black banded shrimp) contained significantly less total carotenoid (4.3-7 ppm). The only major carotenoid being astaxanthin. 4. Commercially available diets contained only trace quantities of canthaxanthin. 5. Nutritional deficiency with respect to carotenoids is suggested as the cause of blue disease in farmed P. monodon.

Topics: Animals; beta Carotene; Carotenoids; Penaeidae; Xanthophylls

The quantum yield for the photobleaching of astaxanthin (the carotenoid of wild salmonoids) and of canthaxanthin (the closely related carotenoid used as a feeding additive for farmed salmonoids) has been determined for monochromatic light at different wavelengths and in different solvents. Astaxanthin is less sensitive to light than canthaxanthin. The photobleaching is strongly wavelength dependent, and the quantum yield for astaxanthin dissolved in chloroform at 22 degrees C is 3.2 x 10(-1) mol.Einstein-1 at 254 nm, 3.1 x 10(-2) at 313 nm, and 1.6 x 10(-6) at 436 nm, respectively. The quantum yields are less dependent on the nature of the solvent and show no simple correlation with oxygen solubility, i.e. for 366 nm excitation of astaxanthin the quantum yields are 6.1 x 10(-5) mol.Einstein-1 in acetone, 1.2 x 10(-4) in saturated vegetable oil, 1.9 x 10(-4) in chloroform, and 3.4 x 10(-4) solubilized in water, respectively. The photobleaching quantum yield provides an objective measure of the light sensitivity of the carotenoids in relation to the discolouration of carotenoid-pigmented salmonoids. The quantum yield was also found to be independent of the carotenoid concentration and, in a homogenous solution, of light intensities. For astaxanthin solubilized in water, the quantum yield increases for low light intensities. Excitation of astaxanthin solubilized in water using visible light shows that the photobleaching quantum yield is independent of temperature, while excitation at 313 nm shows an increase in the quantum yield with increasing temperatures, corresponding to an energy of activation of 28 kJ.mol-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; beta Carotene; Canthaxanthin; Carotenoids; Food Handling; Food Irradiation; Food Preservation; Light; Salmon; Salmonidae; Solubility; Trout; Xanthophylls

The complete sequence has been determined for the C1 subunit of crustacyanin, an astaxanthin-binding protein from the carapace of the lobster Homarus gammarus (L.). The polypeptide, 181 residues long, is similar (38% identity) to the other main subunit, A2 and to plasma retinol-binding protein. The tertiary structure of the C1 subunit has been modelled on that derived for the A2 subunit from the coordinates of retinol-binding protein. Residues lining the putative binding cavities and at the putative carotenoid binding sites of the two subunits are highly conserved. The carotenoid environments are characterized by a preponderance of aromatic and polar residues and the absence of charged side-chains. A tentative model for the dimer, beta-crustacyanin, formed between the two subunits with their associated carotenoid ligands, is discussed. The model is based on the crystal structure of the dimer of bilin-binding protein, a member of the same superfamily. This structure has enabled us to examine mechanisms for the bathochromic spectral shift of the protein-bound carotenoid and to identify likely contact regions between dimers in octameric alpha-crustacyanin.

Topics: Amino Acid Sequence; Animals; beta Carotene; Binding Sites; Carotenoids; Carrier Proteins; Chemical Phenomena; Chemistry, Physical; Macromolecular Substances; Models, Molecular; Molecular Sequence Data; Nephropidae; Protein Conformation; Proteins; Xanthophylls

Crustacyanin, cross-linked with dimethyl pimelimidate to stabilize the protein against denaturation, was used to test the effects of tryptophan modification with BNPS-skatole [3-bromo-3-methyl-2-(nitrophenylmercaptol)-3H-indole] on the ability of the apoprotein to recombine with astaxanthin. The cross-linked apoprotein re-forms alpha-crustacyanin with astaxanthin in reasonable yield following incubation of the protein under the conditions for tryptophan modification in the absence of BNPS-skatole. The BNPS-skatole-treated protein reconstitutes with astaxanthin to give a carotenoprotein with lambda max. at 472 nm, that of the carotenoid in hexane, in a yield similar to that of the BNPS-skatole-untreated control. The implied involvement of tryptophan residues at the sites of astaxanthin attachment in crustacyanin and their possible roles in the binding sites of vitamin A in vitamin A-proteins are discussed in relation to the bathochromic spectral shifts of the chromophores.

Topics: Animals; Apoproteins; beta Carotene; Binding Sites; Carotenoids; Carrier Proteins; Electrophoresis, Polyacrylamide Gel; Models, Molecular; Nephropidae; Protein Conformation; Proteins; Skatole; Spectrophotometry; Tryptophan; Xanthophylls

The value of astaxanthin, a carotenoid pigment, in the treatment of oxidative injury is assessed. Astaxanthin protects the mitochondria of vitamin E-deficient rats from damage by Fe2(+)-catalyzed lipid peroxidation both in vivo and in vitro. The inhibitory effect of astaxanthin on mitochondrial lipid peroxidation is stronger than that of alpha-tocopherol. Thin layer chromatographic analysis shows that the change in phospholipid components of erythrocytes from vitamin E-deficient rats induced by Fe2+ and Fe3(+)-xanthine/xanthine oxidase system was significantly suppressed by astaxanthin. Carrageenan-induced inflammation of the paw is also significantly inhibited by administration of astaxanthin. These data indicate that astaxanthin functions as a potent antioxidant both in vivo and in vitro.

Topics: Animals; beta Carotene; Carotenoids; Erythrocyte Membrane; Iron; Kinetics; Lipid Peroxidation; Male; Mitochondria, Liver; Oxidative Phosphorylation; Rats; Rats, Inbred Strains; Reference Values; Vitamin E; Vitamin E Deficiency; Xanthophylls

The quality of wild salmon and farmed rainbow trout from aquaculture, both packed in transparent vacuum-skin packaging, was followed during storage for 6 months in an illuminated freezer cabinet (product temperature -17 degrees C, half of the packs protected against light, and half of the packs fully exposed to light), combining (a) colour determination of the carotenoid-pigment flesh by tristimulus colorimetry, (b) determination of thiobarbituric-acid-reactive substances (TBA value), (c) carotenoid analysis and, at the end of the storage experiment, (d) sensory evaluation. Rancidity developed faster in steaks of wild salmon (TBA increased during 6 months of storage from 2.8 mumols malonaldehyde/kg flesh to 12.5 mumols/kg for light-protected packages, and to 17.6 mumols/kg for packages exposed to fluorescent light) as compared to steaks of farmed rainbow trout (TBA increased from 1.2 to 5.8 mumols/kg, independent of light exposure), a finding also confirmed by sensory evaluation. In both products, the carotenoid pigment was identified as astaxanthin; salmon steaks, the product more susceptible to developing rancidity, had the lower astaxanthin content (rainbow trout 9.1 mg/kg flesh, salmon 4.9 mg/kg, prior to storage). While the astaxanthin content remained virtually constant in salmon steaks during storage, the content decreased significantly in steaks of rainbow trout, an observation which suggests the role of astaxanthin as a sacrificial protector against radical processes.

Topics: Animals; beta Carotene; Carotenoids; Chromatography, High Pressure Liquid; Cryopreservation; Food Analysis; Food Preservation; Light; Salmon; Trout; Xanthophylls

The apoproteins of the lobster carotenoprotein, crustacyanin, show single high-affinity binding sites for the hydrophobic fluorescence probes 8-anilo-1-naphthalenesulphonic acid and cis-parinaric acid, and exhibit fluorescence transfer from tryptophan to the ligands. These results, together with information from the amino acid sequences, infer that the native carotenoid, astaxanthin, is bound to each apoprotein within an internal hydrophobic pocket, or calyx.

Topics: Animals; beta Carotene; Binding Sites; Carotenoids; Carrier Proteins; Fluorescent Dyes; Ligands; Nephropidae; Proteins; Xanthophylls

The carotenoid esters of the shrimp, Penaeus schmitti, were investigated by thin layer chromatography and absorption spectrophotometry. Astaxanthin monoester and zeaxanthin monoester were identified in hepatopancreas and ovaries during the sexual development. The nature of fatty acids derived from these natural esters has been determined quantitatively by gas chromatography of their methyl esters. The variations of linkage between fatty acids and carotenoids during ovogenesis are measured. The role of zeaxanthin monoester in carotenoids transfer from hepatopancreas to ovaries during this sexual development, and relations between lipid and carotenoid metabolism are discussed.

Topics: Animals; beta Carotene; Carotenoids; Fatty Acids; Female; Liver; Ovary; Pancreas; Penaeidae; Xanthophylls; Zeaxanthins

Two reconstituted carotenoproteins have been studied by resonance Raman spectroscopy. They were prepared from the apoprotein of the Asterias rubens carotenoprotein, asteriarubin and either astaxanthin or 15,15'-didehydroastaxanthin. Spectral properties of dehydrocarotenoids are first discussed. The spectral properties of the complexes are compared to those of the free carotenoids and of other carotenoproteins containing astaxanthin, and possible protein-carotenoid interactions are discussed. Greater delocalisation of the pi-electron system in the central part of the polyene chain, and the role of lateral methyl groups in binding is emphasised.

Topics: Animals; Apoproteins; beta Carotene; Carotenoids; Glycoproteins; Molecular Conformation; Protein Binding; Proteins; Spectrum Analysis, Raman; Starfish; Xanthophylls

The collapse pressure at the air-water interface of monomolecular films of 1,2-distearoyl derivatives of phosphatidylcholine and digalactosyldiacylglycerol containing various proportions of the carotenoid astaxanthin was related to the composition of the monolayer. The results were analysed by using a regular-association approximation by which it is assumed that there is a stepwise formation of ABi-type associations where A and B represent the diacyl lipid and astaxanthin respectively and 1 less than or equal to i less than or equal to 6. This treatment provides an adequate description of the experimental data and permits calculation of equilibrium constants for the steps in complex-formation; each step is said to have the same equilibrium constant. The value for the collapse-surface-pressure increment associated with formation of ABi complexes was also derived. Calculated values of equilibrium constants and collapse-surface-pressure increments are greater for phosphatidylcholine/astaxanthin mixed monolayers than for digalactosyldiacylglycerol/astaxanthin mixed monolayers. These differences are discussed in terms of intermolecular interactions between components in the two systems.

Topics: beta Carotene; Carotenoids; Galactolipids; Glycolipids; Macromolecular Substances; Membrane Lipids; Models, Biological; Phosphatidylcholines; Pressure; Surface Properties; Thermodynamics; Xanthophylls

1. Based on the contents and individual composition of carotenoids in the muscle, serum and ovaries of chum salmon during spawning migration, the reductive metabolism of astaxanthin to zeaxanthin was presumed to take place in the muscle of both male and female. 2. The metabolic rates of zeaxanthin and 4-keto-zeaxanthin in female serum were much faster than those in male serum.

Topics: Animals; beta Carotene; Carotenoids; Chromatography, High Pressure Liquid; Female; Male; Muscles; Ovary; Salmon; Xanthophylls; Zeaxanthins

In rainbow trout (Salmo gairdneri), the dietary astaxanthin diesters were mostly absorbed and accumulated in their integuments keeping their configurations, and partially metabolized to (3R, 3'R)-zeaxanthin (major) and/or (3R, 3'S)-zeaxanthin (medium) and/or (3S,3'S)-zeaxanthin (minor). In tilapia (Tilapia nilotica), the three stereoisomers of astaxanthin diesters were promptly metabolized to only (3S,3'S)-astaxanthin, and subsequently to (3R,3'R)-zeaxanthin and/or (3R,3'S)-zeaxanthin and/or (3S,3'S)-zeaxanthin at an invariable ratio, 4:1:0.3. The above facts indicate that the conversion from 3S- to 3R-configuration was carried out in vivo, and vice versa, and that astaxanthins were reductively metabolized to zeaxanthins in both the fish.

Topics: Animals; beta Carotene; Carotenoids; Fishes; Salmonidae; Species Specificity; Stereoisomerism; Trout; Xanthophylls; Zeaxanthins

An orange carotenoprotein (lambda max = 480 nm) containing astaxanthin as prosthetic group was extracted and purified from the carapace of the crab Macropipus puber. The extraction was made possible by means of Triton X-100, yielding an orange carotenoprotein, with a molecular weight of about 56,000. Sodium dodecyl sulfate polyacrylamide gel electrophoresis indicated only a single polypeptide of 14,000 daltons, suggesting that the orange caroteno-protein was a tetrameric form.

Topics: Animals; beta Carotene; Brachyura; Carotenoids; Chromatography, Gel; Electrophoresis, Polyacrylamide Gel; Lipids; Molecular Weight; Pigments, Biological; Proteins; Spectrophotometry; Xanthophylls

The circular dichroic (CD) spectra of the alpha-, beta'-, and gamma-crustacyanins, ovoverdin, and the yellow lobster-shell protein were measured in the region 200-750 nm, for comparison with the CD spectrum of the free carotenoid astaxanthin. The two carotenoid chromophores of ovoverdin gave a CD spectrum with a series of bands of alternating sign and ellipticities up to 1.9 X 10(5) degree X cm2 X dmol-1, comparable to the low temperature CD spectrum of astaxanthin in the UV region. The visible region of ovoverdin also contained strong CD bands where astaxanthin itself has very weak ones. The blue (640 nm) chromophore of ovoverdin gave a broad negative CD feature quite different from the blue chromophores of the three crustacyanins. The crustacyanins have a broad positive feature between 400 and 610 nm with ellipticities up to 2 X 10(5) degree X cm2 X dmol-1, followed at higher wavelengths by a negative band with an ellipticity up to 1 X 10(5) degree X cm2 X dmol-1. Gaussian curve-fitting procedures showed the positive features to consist of a minimum of two or three CD bands. In addition to the exciton bands at the main visible absorption, the yellow lobster-shell protein had a pair of CD bands of equal but opposite ellipticity associated with an absorption band between 250 and 340 nm. The UV regions of the CD spectra of the five carotenoproteins have bands from both carotenoid and protein chromophores, and possible assignments of these bands to one or the other of the two types of chromophore are proposed.

Topics: Animals; beta Carotene; Carotenoids; Carrier Proteins; Circular Dichroism; Nephropidae; Proteins; Temperature; Xanthophylls

Topics: Animals; beta Carotene; Carotenoids; Crustacea; Proteins; Xanthophylls

alpha-Crustacyanin exhibits two CD extrema with negative and positive bands at 690 and 583 nm in phosphate buffer, respectively. The CD spectrum is interpreted in terms of dipoledipole coupling between the transition moments of the two astaxanthin molecules on each subunit. The CD splitting yields an exciton bandwidth of 2800 cm-1, corresponding to an inter-chromophore distance of ca. 13 A in which the two astaxanthin molecules exist in a dimeric array with a mutual orientation angle of ca. 90 degrees. The SDS denaturation completely abolishes the long wavelength CD splitting, while 2 M NaCl reduces the overall CD intensity without destroying the split CD spectrum. It is suggested that the characteristic CD extrema at 583 and 690 nm arise from the intra-subunit astaxanthin-astaxanthin coupling. The binding site for astaxanthin contains one or more tryptophan residues, as the tryptophan fluorescence is quenched by energy transfer from the tryptophan to the prosthetic group in the native form of the carotenoprotein. The strong red shift (lambda max 487 nm) is probably due to interactions between tryptophan residue(s) and the chromophore molecules through dipole-dipole or partial charge transfer forces.

Topics: Animals; beta Carotene; Carotenoids; Carrier Proteins; Chemical Phenomena; Chemistry; Circular Dichroism; Nephropidae; Protein Denaturation; Proteins; Tryptophan; Xanthophylls

Small amounts of beta-carotene and lutein were found in Orconectes pellucidus pellucidus. Cambarus bartonii tenebrosus from the same cave contained much less carotenoid than surface crayfish. Astaxanthin, the principal carotenoid of most Crustacea, was absent from O. p. pellucidus, but accounted for 83 percent of the carotenoid of C. b. tenebrosus. These findings support other observations that pigmentation is dependent on the amount of carotenoid in the diet rather than on the presence of light. Furthermore, they suggest that O. p. pellucidus has lost or has never developed the ability to oxidize dietary carotenoids.

Topics: Animals; Astacoidea; beta Carotene; Carotenoids; Chemistry Techniques, Analytical; Crustacea; Diet; Feeding Behavior; Lutein; Pigmentation; Research; Xanthophylls

Topics: beta Carotene; Carotenoids; Cyprinodontiformes; Humans; In Vitro Techniques; Intestine, Small; Intestines; Vitamin A; Xanthophylls