Balance of xanthophylls molecular and protonated molecular ions in electrospray ionization.

This paper reports the chemical evidence of the balance between radical molecular ions and protonatedmolecules of xanthophylls (an oxygen-containing carotenoid) with a conjugated pi-system (polyene) and oxygen as a heteroatom in ESI and HRESI mass spectrometry. The ionization energy of neutral xanthophylls was calculated by semi-empirical methods. The results were compared with those previously published for carotenoids and retinoids, which have also been shown in ESI-MS to form M(+*) and [M + H](+), respectively. This study demonstrates, for the first time, the correlation of an extended conjugation and the presence of oxygen in the formation and balance of M(+*) or [M + H](+) for the carotenoids, neoxanthin, lutein, violaxanthin and zeaxanthin.

[Effects of the dietary incorporation of carotenoid pigments on gonad development and oocyte maturity in females of rainbow trout (Oncorhynchus mykiss, Walbaum, 1972)]. / Efectos de la incorporación en la dieta de pigmentos carotenoides, sobre el desarrollo gonadal y la madurez de los ovocitos en hembras de trucha arco iris (Onchorhynchus mykiss, Walbaum, 1972).

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.

Astaxanthin hyperproduction by Phaffia rhodozyma (now Xanthophyllomyces dendrorhous) with raw coconut milk as sole source of energy.

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.

Study of the expression of carotenoid biosynthesis genes in wild-type and deregulated strains of Xanthophyllomyces dendrorhous (Ex.: Phaffia rhodozyma).

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.

Beta-carotene storage, conversion to retinoic acid, and induction of the lipocyte phenotype in hepatic stellate cells.

Hepatic stellate cells (HSCs) are the major site of retinol (ROH) metabolism and storage. GRX is a permanent murine myofibroblastic cell line, derived from HSCs, which can be induced to display the fat-storing phenotype by treatment with retinoids. Little is known about hepatic or serum homeostasis of beta-carotene and retinoic acid (RA), although the direct biogenesis of RA from beta-carotene has been described in enterocytes. The aim of this study was to identify the uptake, metabolism, storage, and release of beta-carotene in HSCs. GRX cells were plated in 25 cm(2) tissue culture flasks, treated during 10 days with 3 micromol/L beta-carotene and subsequently transferred into the standard culture medium. beta-Carotene induced a full cell conversion into the fat-storing phenotype after 10 days. The total cell extracts, cell fractions, and culture medium were analyzed by reverse phase high-performance liquid chromatography for beta-carotene and retinoids. Cells accumulated 27.48 +/- 6.5 pmol/L beta-carotene/10(6) cells, but could not convert it to ROH nor produced retinyl esters (RE). beta-Carotene was directly converted to RA, which was found in total cell extracts and in the nuclear fraction (10.15 +/- 1.23 pmol/L/10(6) cells), promoting the phenotype conversion. After 24-h chase, cells contained 20.15 +/- 1.12 pmol/L beta-carotene/10(6) cells and steadily released beta-carotene into the medium (6.69 +/- 1.75 pmol/ml). We conclude that HSC are the site of the liver beta-carotene storage and release, which can be used for RA production as well as for maintenance of the homeostasis of circulating carotenoids in periods of low dietary uptake.

Antioxidant defense system in the apple snail eggs, the role of ovorubin.

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.

Dietary intake and risk of persistent human papillomavirus (HPV) infection: the Ludwig-McGill HPV Natural History Study.

The association between dietary intake and persistence of type-specific human papillomavirus (HPV) infection, during a 12-month period, among 433 women participating in the Ludwig-McGill HPV Natural History Study was evaluated by use of a nested case-control design. Dietary intake was assessed by a food-frequency questionnaire at the month-4 visit. HPV status was assessed at months 0, 4, 8, and 12 by polymerase chain reaction (MY09/11). Only women who ever tested positive for HPV were included in the present study: 248 had transient HPV infections (1 of 4 positive tests or nonconsecutively positive), and 185 had persistent HPV infections (> or =2 consecutive tests positive for the same HPV type). Risk of type-specific, persistent HPV infection was lower among women reporting intake values of beta-cryptoxanthin and lutein/zeaxanthin in the upper 2 quartiles and intake values of vitamin C in the upper quartile, compared with those reporting intake in the lowest quartile. Consumption of papaya > or =1 time/week was inversely associated with persistent HPV infection.

Optimization of biomass, total carotenoids and astaxanthin production in Haematococcus pluvialis Flotow strain Steptoe (Nevada, USA) under laboratory conditions.

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.

Multinational study of major breast milk carotenoids of healthy mothers.

BACKGROUND: Carotenoids in serum vary between countries and within populations with evidence suggesting a qualitative relationship to diet. Breast milk carotenoids furnish a source of vitamin A and potentially provide immunoprotection and other health benefits for infants. There have been numerous studies of milk carotenoid concentrations in undernourished populations; however, carotenoid concentrations have not previously been compared in populations of well-nourished mothers. AIM OF STUDY: To compare concentrations of five major carotenoid groups: alpha-carotene, beta-carotene, beta-cryptoxanthin, lutein/zeaxanthin, and lycopene in breast milk of healthy women from Australia, Canada, Chile, China, Japan, Mexico, the Philippines, the United Kingdom, and the United States, and to qualitatively compare patterns of dietary intake with milk carotenoid concentrations. METHODS: Breast milk collected from healthy lactating women was analyzed for concentrations of five carotenoids and retinol and quantitated relative to total milk lipid. All determinations were performed in a single research laboratory using standardized methodology. Mothers consumed their usual diets and provided a single 24-h dietary recall. RESULTS: Breast milk carotenoid concentrations varied greatly among countries, with the greatest differences in beta-cryptoxanthin (approximately 9-fold) and the least in alpha-carotene and lycopene (approximately 3-fold). Breast milk retinol concentrations varied approximately 2-fold across countries. The provitamin A carotenoids alpha-carotene, beta-carotene, and beta-cryptoxanthin as a group accounted for > 50 % of the carotenoids measured. Total breast milk carotenoids were highest in Japanese and lowest in Philippine mothers. Breast milk beta-carotene concentrations were highest in Chile and lowest in the Philippines. CONCLUSIONS: Patterns of breast milk carotenoids were unique to each country and qualitative patterns reflected the dietary carotenoid supply.

Frequent intake of tropical fruits that are rich in beta-cryptoxanthin is associated with higher plasma beta-cryptoxanthin concentrations in Costa Rican adolescents.

Dietary tocopherols and carotenoids may play a role in preventing cancer and cardiovascular diseases. Because these may begin to develop during adolescence, dietary patterns during this period could influence long-term risk. The objective of this study was to examine the intake and plasma concentrations of the major carotenoids and tocopherols in 159 adolescents (mean +/- SD, 15.5 +/- 2.5 y old) living in Costa Rica. All participants completed a 135-item food-frequency questionnaire and provided a fasting blood sample. Carotenoid and tocopherol intakes were adjusted for total energy and plasma concentrations for total cholesterol. The relative abundance of carotenoids in the diet was similar to their distribution in plasma; lycopene was the most abundant, followed by beta-carotene and lutein + zeaxanthin. gamma-Tocopherol was more abundant than alpha-tocopherol in the diet, but alpha-tocopherol was approximately sevenfold higher in plasma. The highest diet-plasma correlations (adjusted for age, sex and body mass index) were 0.38 for beta-cryptoxanthin, 0.33 for gamma-tocopherol and 0.17 for lutein + zeaxanthin (all P < 0.05). All other correlations were r < 0.15. Papaya intake was the best food predictor of plasma beta-cryptoxanthin concentrations (r = 0.41). Subjects that frequently (> or =3/d) consumed tropical fruits with at least 50 micro g/100 g beta-cryptoxanthin (papaya, tangerine, orange and watermelon) had twofold the plasma beta-cryptoxanthin concentrations of those with intakes of <4/wk (P for trend = 0.0009). In sum, the diet-plasma carotenoid and tocopherol correlations were generally low in Costa Rican adolescents. Intakes of beta-cryptoxanthin and papaya, a tropical fruit frequently consumed in Latin America, were the best predictors of beta-cryptoxanthin concentrations in plasma.

Individual carotenoid concentrations in adipose tissue and plasma as biomarkers of dietary intake.

BACKGROUND: Plasma and adipose tissue concentrations of carotenoids are thought to reflect short- and long-term intakes of carotenoids, respectively. The ability of adipose tissue carotenoid concentrations to reflect dietary intake in population studies is unknown. OBJECTIVE: We examined the relation between intakes of the major dietary carotenoids and their concentrations in plasma and adipose tissue. DESIGN: A blood sample and an adipose tissue biopsy sample were collected from 115 women and 344 men in Costa Rica after they had fasted overnight, and a dietary interview based on a 135-item food-frequency questionnaire was administered. After carotenoid intake was adjusted for total energy intake and plasma concentrations were adjusted for HDL-, LDL-, and VLDL-cholesterol concentrations, we calculated partial Spearman correlation coefficients that were adjusted for age, sex, smoking, and body mass index. RESULTS: In women, the correlations (r) between intakes and concentrations of alpha-carotene, beta-carotene, beta-cryptoxanthin, and lutein+zeaxanthin were 0.25, 0.29, 0.44, and 0.17, respectively (P < 0.05 for r > or = 0.19), in adipose tissue and 0.26, 0.13, 0.55, and 0.22 in plasma. In men, these values were 0.04, 0.07, 0.23, and 0.06 in adipose tissue and 0.24, 0.22, 0.44, and 0.20 in plasma. In women and men, correlations for lycopene were higher in plasma (r = 0.19 and 0.35, respectively) than in adipose tissue (r = 0.14 and 0.26). The relative abundance of each carotenoid in the diet was similar to its distribution in plasma but not in adipose tissue. CONCLUSION: The usefulness of adipose tissue and plasma carotenoids as biomarkers of intake is similar, although correlations for individual carotenoids vary substantially.

Optimization of astaxanthin production by Phaffia rhodozyma through factorial design and response surface methodology.

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.

Complementation analysis with new genetic markers in Phaffia rhodozyma.

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.

Genetic transformation of astaxanthin mutants of Phaffia rhodozyma.

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.

[Microbial sources of pigments]. / Fuentes microbianas de pigmentos.

Pigments from natural sources has been obtained since long time ago, and their interest has increased due to the toxicity problems caused by those of synthetic origin. In this way the pigments from microbial sources are a good alternative. Some of more important natural pigments, are the carotenoids, flavonoids (anthocyanins) and some tetrapirroles (chloropyls, phycobilliproteins). Another group less important are the betalains and quinones. The carotenoids are molecules formed by isoprenoids units and the most important used as colorant are the alpha and beta carotene which are precursors of vitamin A, and some xantophylls as astaxanthin. The pigment more used in the industry is the beta-carotene which is obtained from some microalgae and cyanobacteria. The astaxanthin another important carotenoid is a red pigment of great commercial value, and it is used in the pharmaceutical feed and acuaculture industries. This pigments is mainly obtained from Phaffia rhodozyma and Haematococcus pluvialis and other organisms. The phycobilliproteins obtained from cyanobacteria and some group of algae, have recently been increased on the food industries. In the last years it has been used as fluorescent marker in biochemical assays. Our research group have carried out studies about the factors that improve the production of these pigments obtained from different microbial species as well as the methods for their extraction and application.

Genetics and electrophoretic karyotyping of wild-type and astaxanthin mutant strains of Phaffia rhodozyma.

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.

Dietary fiber and risk of breast cancer: a case-control study in Uruguay.

To examine whether dietary fiber modifies breast cancer risk, a case-control study involving 351 newly diagnosed patients with breast cancer and 356 hospitalized controls was conducted in Uruguay. Dietary patterns were assessed in detail by use of a food frequency questionnaire on 64 items, which allowed the calculation of total energy intake. Nutrient residuals were calculated through regression analysis. After adjustment for potential confounders (which included age, residence, family history of breast cancer, prior history of benign breast disease, parity, total energy, red meat, lutein/zeaxanthin and quercetin intake, and menopausal status), dietary fiber and total nonstarch polysaccharides were associated with a strong reduction in risk of breast cancer (odds ratio for uppermost quartile of total dietary fiber = 0.51, 95% confidence limit = 0.31-0.82). Also the dose-response pattern was highly significant (p < 0.001). The inverse association was observed in pre- and post-menopausal women and was similar for soluble and insoluble fiber. Furthermore, dietary fiber displayed a strong joint effect with fat, quercetin, and lutein/zeaxanthin.

Pigmentation of the rainbow trout (Oncorhynchus mykiss) with oil-extracted astaxanthin from the langostilla (Pleuroncodes planipes).

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).

Serum carotenoid concentrations and their reproducibility in children in Belize.

Suggestions that carotenoid-containing foods are beneficial in maintaining health have led to several studies of circulating carotenoid concentrations of adults. Because few data are available for children, we report serum carotenoid concentrations of 493 children in Belize. Carotenoid concentrations were determined as part of a survey of vitamin A status of children, most between 65 and 89 mo of age. Reproducibility was tested by collecting a second blood sample 2 wk after the first collection from a subset of children (n = 23) who consumed their habitual diet with no treatment during the interim. Predominant serum carotenoids were lutein/zeaxanthin and beta-carotene, which accounted for 26% and 24% of median total carotenoids, respectively. The three provitamin A carotenoids, alpha- and beta-carotene and beta-cryptoxanthin, constituted 51% of median total carotenoid concentrations. Partial correlations of each carotenoid with fasting retinol concentration indicated that beta-carotene had the highest correlation. Concordance correlation coefficients (rc) for fasting carotenoid concentrations determined 2 wk apart were > or = 0.89 for lycopene, beta-cryptoxanthin, and alpha- and beta-carotene. The rc for lutein/zeaxanthin and total carotenoids was lower, 0.59 and 0.68, respectively, because of higher lutein/zeaxanthin concentrations at the second sampling than at the first. The reproducibility of the concentrations suggests both that individuals have characteristic profiles and that serum carotenoid concentrations can be measured randomly over > or = 2 wk without significant bias.

[Bioassay of rainbow trout (Oncorhynchus mykiss) pigmentation using ancho chili (Capsicum annuum) extracts]. / Bioensayo de pigmentación de trucha arcoiris (Oncorhynchus mykiss) con extractos de chile ancho (Capsicum annuum).

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.