Dietary supplementation with a natural carotenoid mixture decreases oxidative stress

Objective: To determine whether dietary supplementation with a natural carotenoid mixture counteracts the enhancement of oxidative stress induced by consumption of fish oil. Design: A randomised double-blind crossover dietary intervention. Setting: Hugh Sinclair Unit of Human Nutrition, School of Food Biosciences, The University of Reading, Whiteknights PO Box 226, Reading RG6 6AP, UK. Subjects and intervention: A total of 32 free-living healthy nonsmoking volunteers were recruited by posters and e-mails in The University of Reading. One volunteer withdrew during the study. The volunteers consumed a daily supplement comprising capsules containing fish oil (4 x 1 g) or fish oil (4 x 1 g) containing a natural carotenoid mixture (4 x 7.6 mg) for 3 weeks in a randomised crossover design separated by a 12 week washout phase. The carotenoid mixture provided a daily intake of beta-carotene (6.0 mg), alpha-carotene (1.4 mg), lycopene (4.5 mg), bixin (11.7 mg), lutein (4.4 mg) and paprika carotenoids (2.2 mg). Blood and urine samples were collected on days 0 and 21 of each dietary period. Results: The carotenoid mixture reduced the fall in ex vivo oxidative stability of low-density lipoprotein (LDL) induced by the fish oil (P = 0.045) and it reduced the extent of DNA damage assessed by the concentration of 8-hydroxy-2'-deoxyguanosine in urine (P = 0.005). There was no effect on the oxidative stability of plasma ex vivo assessed by the oxygen radical absorbance capacity test. beta- Carotene, alpha-carotene, lycopene and lutein were increased in the plasma of subjects consuming the carotenoid mixture. Plasma triglyceride levels were reduced significantly more than the reduction for the fish oil control (P = 0.035), but total cholesterol, HDL and LDL levels were not significantly changed by the consumption of the carotenoid mixture. Conclusions: Consumption of the natural carotenoid mixture lowered the increase in oxidative stress induced by the fish oil as assessed by ex vivo oxidative stability of LDL and DNA degradation product in urine. The carotenoid mixture also enhanced the plasma triglyceride-lowering effect of the fish oil.

A novel combined biomarker including plasma carotenoids, vitamin C, and ferric reducing antioxidant power is more strongly associated with fruit and vegetable intake than the individual components

BACKGROUND: Monitoring of fruit and vegetable (F&V) intake is fraught with difficulties. Available dietary assessment methods are associated with considerable error, and the use of biomarkers offers an attractive alternative. Few studies to date have examined the use of plasma biomarkers to monitor or predict the F&V intake of volunteers consuming a wide range of intakes from both habitual F&V and manipulated diets. OBJECTIVE: This study tested the hypothesis that an integrated biomarker calculated from a combination of plasma vitamin C, cholesterol-adjusted carotenoid concentration and Ferric Reducing Antioxidant Power (FRAP) had more power to predict F&V intake than each individual biomarker. METHODS: Data from a randomized controlled dietary intervention study [FLAVURS (Flavonoids University of Reading Study); n = 154] in which the test groups observed sequential increases of 2.3, 3.2, and 4.2 portions of F&Vs every 6 wk across an 18-wk period were used in this study. RESULTS: An integrated plasma biomarker was devised that included plasma vitamin C, total cholesterol-adjusted carotenoids, and FRAP values, which better correlated with F&V intake (r = 0.47, P < 0.001) than the individual biomarkers (r = 0.33, P < 0.01; r = 0.37, P < 0.001; and r = 0.14, respectively; P = 0.099). Inclusion of urinary potassium concentration did not significantly improve the correlation. The integrated plasma biomarker predicted F&V intake more accurately than did plasma total cholesterol-adjusted carotenoid concentration, with the difference being significant at visit 2 (P < 0.001) and with a tendency to be significant at visit 1 (P = 0.07). CONCLUSION: Either plasma total cholesterol-adjusted carotenoid concentration or the integrated biomarker could be used to distinguish between high- and moderate-F&V consumers. This trial was registered at www.controlled-trials.com as ISRCTN47748735.

Carotenoids but not flavonoids are associated with improvements in spatial working memory in younger adults in a flavonoid-rich v. -poor fruit and vegetable intervention study

Findings from animal studies suggest that components of fruit and vegetables (F&V) may protect against, and even reverse, age-related decline(1,2) in aspects of cognitive functioning such as spatial working memory (SWM). Human subjects in vivo and in vitro studies indicate that anti-inflammatory, anti-oxidant and cell-signalling properties of flavonoids and carotenoids, non-nutrient components of F&V, may underpin this protective effect(3–5). The Flavonoid University of Reading Study (FLAVURS), designed to explore the dose-response relationship between dietary F&V flavonoids and CVD, enabled the investigation of such an association with SWM. FLAVURS is an 18-week parallel three-arm randomised controlled dietary intervention trial with four time points, measured at 6-weekly intervals from baseline. Low F&V consumers at risk of CVD aged 26–70 years were randomly assigned to high flavonoid (HF), low flavonoid (LF) or control group. F&V intake increased by two daily 80 g portions every 6 weeks, with either HF or LF F&V, in addition to each participant's habitual diet, while controls maintained their habitual diet. At each visit, participants completed a cognitive test battery with SWM as the primary outcome. The HF group showed significantly higher levels of urinary flavonoids than LF or controls at 12 weeks (P<0.001) as expected, but surprisingly only higher levels than LF at 18 weeks (P<0.01). The LF group showed higher levels of plasma carotenoids than the other groups at 18 weeks (P<0.001). No group differences were found for SWM overall, however, age-group sub-analyses (26–50 and 51–70 years of age) showed differences from 0 to 18 weeks for younger adults, with LF improving significantly more than the other two groups on SWM (P<0.05). As nutritional absorption is known to decrease with age, separate stepwise regressions were performed on the two age groups irrespective of dietary group, with urinary flavonoids and plasma carotenoids as predictors. For younger adults, improved SWM performance from 0 to 18 weeks was associated with higher carotenoid levels, β=0.28, t(55)=2.10, P<0.05, accounting for 7.5% of the variance, R2=0.075, F(1,54)=4.41, P=0.040. For older adults, no between-group SWM differences were found. Findings suggest that F&V-based flavonoids and carotenoids may provide benefits for cognitive function, and that carotenoids in particular may improve cognitive performance in SWM. Given that these benefits were restricted to younger adults, future work is needed to test the reliability of this finding, as well as determine the mechanisms by which age-dependent differences in F&V responsiveness occur.

Supercritical CO(2) extraction of carotenoids from pitanga fruits (Eugenia uniflora L.)

Supercritical carbon dioxide (SC-CO(2)) extraction was employed to extract carotenoids from the freeze-dried pulp of pitanga fruits (Eugenia uniflora L.), an exotic fruit, rich in carotenoids and still little explored commercially. The SC-CO(2) extraction was carried out at two temperatures, 40 and 60 degrees C, and seven pressures, 100, 150, 200, 250, 300, 350 and 400 bar. The carotenoids were determined by high-performance liquid chromatography connected to photodiode array and mass spectrometry detectors. Lycopene, rubixanthin and P-cryptoxanthin were the main carotenoids present in the freeze-dried pitanga pulp, whereas beta-cryptoxanthin concentration was negligible in the SC-CO(2) extracts, for all the investigated state conditions. The maximum recovery of carotenoids was obtained at 60 degrees C and 250 bar, extracting 55% of the total carotenoid content, 74% of the rubixanthin and 78% of the lycopene from the pulp. Under these state conditions, the total carotenoid concentration in the extract was 5474 mu g/g, represented by 66% lycopene and 32% rubixanthin. The experimental state conditions produced different SC-CO(2) extracts with respect to the extraction yield and concentration of different carotenoids, indicating that the supercritical carbon dioxide was selective in the extraction of the pitanga carotenoids as a function of temperature and pressure. (C) 2008 Elsevier B.V. All rights reserved.

Validation of a food-frequency questionnaire assessment of carotenoid and vitamin E intake using weighed food records and plasma biomarkers: the method of triads model.

Background: Reliability or validity studies are important for the evaluation of measurement error in dietary assessment methods. An approach to validation known as the method of triads uses triangulation techniques to calculate the validity coefficient of a food-frequency questionnaire (FFQ).

Objective: To assess the validity of an FFQ estimates of carotenoid and vitamin E intake against serum biomarker measurements and weighed food records (WFRs), by applying the method of triads. Design: The study population was a sub-sample of adult participants in a randomised controlled trial of b-carotene and sunscreen in the prevention of skin cancer. Dietary intake was assessed by a self-administered FFQ and a WFR. Nonfasting blood samples were collected and plasma analysed for five carotenoids (a-carotene, b-carotene, b-cryptoxanthin, lutein, lycopene) and vitamin E. Correlation coefficients were calculated between each of the dietary methods and the validity coefficient was calculated using
the method of triads. The 95% confidence intervals for the validity coefficients were estimated using bootstrap sampling.

Results: The validity coefficients of the FFQ were highest for a-carotene (0.85) and lycopene (0.62), followed by b-carotene (0.55) and total carotenoids (0.55), while the lowest validity coefficient was for lutein (0.19). The method of triads could not be used for b-cryptoxanthin and vitamin E, as one of the three underlying correlations was negative.

Conclusions: Results were similar to other studies of validity using biomarkers and the method of triads. For many dietary factors, the upper limit of the validity coefficients was less than 0.5 and therefore only strong relationships between dietary exposure and disease will be detected.

Evaluation of brief dietary questions to estimate vegetable and fruit consumption - using serum carotenoids and red-cell folate

Objective To evaluate responses to self-administered brief questions regarding consumption of vegetables and fruit by comparison with blood levels of serum carotenoids and red-cell folate.

Design A cross-sectional study in which participants reported their usual intake of fruit and vegetables in servings per day, and serum levels of five carotenoids (α-carotene, β-carotene, β-cryptoxanthin, lutein/zeaxanthin and lycopene) and red-cell folate were measured. Serum carotenoid levels were determined by high-performance liquid chromatography, and red-cell folate by an automated immunoassay system.

Settings and subjects Between October and December 2000, a sample of 1598 adults aged 25 years and over, from six randomly selected urban centres in Queensland, Australia, were examined as part of a national study conducted to determine the prevalence of diabetes and associated cardiovascular risk factors.

Results Statistically significant (P<0.01) associations with vegetable and fruit intake (categorised into groups: ≤1 serving, 2–3 servings and ≥4 servings per day) were observed for α-carotene, β-carotene, β-cryptoxanthin, lutein/zeaxanthin and red-cell folate. The mean level of these carotenoids and of red-cell folate increased with increasing frequency of reported servings of vegetables and fruit, both before and after adjusting for potential confounding factors. A significant association with lycopene was observed only for vegetable intake before adjusting for confounders.

Conclusions These data indicate that brief questions may be a simple and valuable tool for monitoring vegetable and fruit intake in this population.

Effect of carotenoids and background colour on the skin pigmentation of Australian snapper Pagrus auratus (Bloch & Schneider, 1801)

Three 2-factor experiments were conducted to determine the effects of background colour and synthetic carotenoids on the skin colour of Australian snapper Pagrus auratus. Initially, we evaluated the effects on skin colour of supplementing diets for 50 days with 60 mg kg⁻¹ of either astaxanthin (LP; Lucantin^®Pink), canthaxanthin (LR; Lucantin^® Red), apocarotenoic acid ethyl ester (LY; Lucantin^® Yellow), selected combinations of the above or no carotenoids and holding snapper (mean weight=88 g) in either white or black cages. In a second experiment, all snapper (mean weight=142 g) from Experiment 1 were transferred from black to white, or white to white cages to measure the short-term effects of cage colour on skin L*, a* and b* colour values. Skin colour was measured after 7 and 14 days, and total carotenoid concentrations were determined after 14 days.

Cage colour was the dominant factor affecting the skin lightness of snapper with fish from white cages much lighter than fish from black cages. Diets containing astaxanthin conferred greatest skin pigmentation and there were no differences in redness (a*) and yellowness (b*) values between snapper fed 30 or 60 mg astaxanthin kg⁻¹. Snapper fed astaxanthin in white cages displayed greater skin yellowness than those in black cages. Transferring snapper from black to white cages increased skin lightness but was not as effective as growing snapper in white cages for the entire duration. Snapper fed astaxanthin diets and transferred from black to white cages were less yellow than those transferred from white to white cages despite the improvement in skin lightness (L*), and the total carotenoid concentration of the skin of fish fed astaxanthin diets was lower in white cages. Diets containing canthaxanthin led to a low level of deposition in the skin while apocarotenoic acid ethyl ester did not alter total skin carotenoid content or skin colour values in snapper.

In a third experiment, we examined the effects of dietary astaxanthin (diets had 60 mg astaxanthin kg⁻¹ or no added carotenoids) and cage colour (black, white, red or blue) on skin colour of snapper (mean weight=88 g) after 50 days. Snapper fed the astaxanthin diet were more yellow when held in red or white cages compared with fish held in black or blue cages despite similar feed intake and growth. The skin lightness (L* values) was correlated with cage L* values, with the lightest fish obtained from white cages. The results of this study suggest that snapper should be fed 30 mg astaxanthin kg⁻¹ in white cages for 50 days to increase lightness and the red colouration prized in Australian markets.

Critical assessment of various techniques for the extraction of carotenoids and co-enzyme Q10 from the Thraustochytrid strain ONC-T18

A variety of techniques for extracting carotenoids from the marine Thraustochytrium sp. ONC-T18 was compared. Specifically, the organic solvents acetone, ethyl acetate, and petroleum ether were tested, along with direct and indirect ultrasonic assisted extraction (probe vs bath) methods. Techniques that used petroleum ether/acetone/water (15:75:10, v/v/v) with 3 h of agitation, or 5 min in an ultrasonic bath, produced the highest extraction yields of total carotenoids (29−30.5 μg g^-1). Concentrations up to 11.5 μg g^-1 of canthaxanthin and 17.5 μg g^-1 of β-carotene were detected in extracts stored for 6 weeks. Astaxanthin and echinenone were also detected as minor compounds. Extracts with and without antioxidants showed similar carotenoid concentration profiles. However, total carotenoid concentrations were approximately 8% higher when antioxidants were used. Finally, an easy-to-perform and inexpensive method to detect co-enzymes in ONC-T18 was also developed using silica gel TLC plates. Five percent methanol in toluene as a mobile phase consistently eluted co-enzyme Q10 standards and could separate the co-enzyme fractions present in ONC-T18.

The combined effect of light intensity and temperature on the chlorophyll and carotenoid pigments of phytoplankton

Chlorophyll a and other photosynthetic pigments are used as indicators of phytoplankton biomass, composition and physiological state. Extraction and HPLC procedures were developed to analyse for chlorophyll and carotenoid pigments. The effect of the environment on pigment production must be quantified before the pigments can be used to accurately estimate biomass or quantitatively describe phytoplankton composition.

Does female nuptial coloration reflect egg carotenoids and clutch quality in the Two-Spotted Goby (Gobiusculus flavescens, Gobiidae)?

Summary

1 - Carotenoid-based ornamentation has often been suggested to signal mate quality, and species with such ornaments have frequently been used in studies of sexual selection.

2 - Female Gobiusculus flavescens (Two-Spotted Goby) develop colourful orange bellies during the breeding season. Belly coloration varies among mature females, and previous work has shown that nest-holding males prefer females with more colourful bellies. Because males invest heavily in offspring during incubation, the evolution of this preference can be explained if colourful females provide males with eggs of higher quality.

3 - We tested this hypothesis by allowing males to spawn with ‘colourful’ and ‘drab’ females and comparing parameters including egg carotenoid concentration, clutch size, hatchability and larval viability between groups. We also investigated relationships between egg carotenoid concentration and clutch quality parameters.

4 - Eggs from colourful females had significantly higher concentrations of total carotenoids than drab females, and photographically quantified belly coloration was a good predictor of egg carotenoid concentration.

5 - Colourful females produced slightly larger clutches, but female belly coloration was not related to any measure of clutch quality. In addition, there were no significant relationships between egg carotenoids and clutch quality. Females with high levels of egg carotenoids spawned slightly earlier, however, possibly because they were more ready to spawn or because of male mate choice.

6 - Our results call into question the generality of a causal link between egg carotenoids and offspring quality.

Fermentation of omega-3 and carotenoid producing marine microorganisms

 The objective of the thesis was to understand and develop the process of fermentation in marine microorganisms for the production of omega-3 fatty acids and carotenoids. Among marine microorganisms, thraustochytrids that belong to phytoplankton group was identified as sources of omega-3 fatty acids and other valuable co-products. In this research, more efficient and cost-effective production of omega-3 oils and other value added products was discussed by addressing the below key objectives. Fermentation strategy using lower cost raw materials, particularly carbon. Screening for new strains that can naturally produce high levels of PUFAs together with useful co-products that can be harvested along with the omega-3 oil.

Testing the interactive effects of carotenoids and polyunsaturated fatty acids on ejaculate traits in the guppy Poecilia reticulata (Pisces: Poeciliidae)

Using the polyandrous livebearing guppy Poecilia reticulata, this study revealed no main effects of carotenoids in the diet on ejaculate traits, but significant main effects of polyunsaturated fatty acids (PUFAs) on sperm viability and weak but significant interacting effects of both nutrients on sperm length. Collectively, these findings not only add evidence that PUFAs are critical determinants of sperm quality, but also provide tentative evidence that for some traits these effects may be moderated by carotenoid intake.

Carotenoid production by Rhodotorula rubra cultivated in sugarcane juice, molasses, and syrup

A produção de biomassa e de carotenoides por Rhodotorula rubra foi estudada em meios à base de caldo, melaço e xarope de cana. Avaliou-se o efeito da suplementação dos meios com nitrogênio na forma de ureia ou do nutriente comercial Nitrofos KL. O delineamento experimental utilizado foi o inteiramente casualizado, no esquema fatorial 3 × 3, sendo um dos fatores o substrato (caldo, melaço e xarope) e o outro a suplementação (controle, ureia e Nitrofos KL). Os resultados foram submetidos à análise de variância e teste de Tukey a 5% de probabilidade. As maiores produções de massa seca de levedura foram obtidas no meio à base de melaço suplementado com ureia ou Nitrofos KL (15,09 e 14,87 g/L, respectivamente). A produção de carotenoides intracelular foi elevada em todos os meios estudados sem suplementação (0,329 mg/g). Para a produção volumétrica, o melhor meio foi o melaço (2,74 mg/L), enquanto a suplementação com ureia e com Nitrofos KL produziu 2,55 e 2,32 mg/L, respectivamente. Os principais carotenoides produzidos foram toruleno, torularrodina e β-caroteno. No meio à base de caldo de cana sem suplementação, houve o menor consumo de carboidratos, enquanto que o meio com suplementação à base de ureia obteve o maior consumo.

Effect of thermal pasteurization and concentration on carotenoid composition of Brazilian Valencia orange juice

Changes in carotenoid pigment content of Brazilian Valencia orange juices due to thermal pasteurization and concentration were studied. Total carotenoid pigment content loss was not significant after thermal pasteurization and concentration. However, thermal effects on carotenoid pigment contents, especially violaxanthin and lutein, were clearly observed and significant (P < 0.05). Pasteurization reduced the content of violaxanthin by 38% and lutein by 20%. The concentration process resulted in loss of lutein (17%). With the loss of lutein, beta-cryptoxanthin became the major carotenoid in the pasteurized and concentrated juices. The provitarnin A content of the juice (beta-carotene, alpha-carotene and beta-cryptoxanthin) and the amount of zeaxanthin, which are considered to be active against age-related macular degeneration and cataracts, did not significantly decrease after pasteurization and concentration. (c) 2006 Elsevier Ltd. All rights reserved.

Major carotenoid composition of Brazilian Valencia orange juice: Identification and quantification by HPLC

The carotenoid composition of Brazilian Valencia orange juice was determined by open column chromatography (OCC) and high-performance liquid chromatography. Carotenoid pigments were extracted using acetone and saponified using 10% methanolic potassium hydroxide. Sixteen pigments were isolated by OCC and identified as alpha-carotene, zeta-carotene, beta-carotene, alpha-cryptoxanthin, beta-cryptoxanthin, lutein-5,6-epoxide, violaxanthin, lutein, antheraxanthin, zeaxanthin, luteoxanthin A, luteoxanthin B, mutatoxanthin A, mutatoxanthin B, auroxanthin B and trollichrome B. Thirteen carotenoid pigments were separated using a ternary gradient (acetonitrile-methanol-ethyl acetate) elution on a C-18 reversed-phase column. Among these, violaxanthin, lutein, zeaxanthin, beta-cryptoxanthin, zeta-carotene, alpha-carotene, and beta-carotene were quantified. The total carotenoid content was 12 +/- 6.7 mg/1, and the major carotenoids were lutein (23%), beta-cryptoxanthin (21%), and zeaxanthin (20%). 2005 Elsevier Ltd. All rights reserved.