Improving the nutritional value of Golden Rice through increased pro-vitamin A content

"Golden Rice" is a variety of rice engineered to produce beta-carotene (pro-vitamin A) to help combat vitamin A deficiency, and it has been predicted that its contribution to alleviating vitamin A deficiency would be substantially improved through even higher beta-carotene content. We hypothesized that the daffodil gene encoding phytoene synthase (psy), one of the two genes used to develop Golden Rice, was the limiting step in beta-carotene accumulation. Through systematic testing of other plant psys, we identified a psy from maize that substantially increased carotenoid accumulation in a model plant system. We went on to develop "Golden Rice 2" introducing this psy in combination with the Erwinia uredovora carotene desaturase (crtI) used to generate the original Golden Rice. We observed an increase in total carotenoids of up to 23-fold (maximum 37 microg/g) compared to the original Golden Rice and a preferential accumulation of beta-carotene.     

Effect of dietary beta-carotene on the accumulation of beta-carotene and vitamin A in plasma and tissues of gilts

The absorption of beta-carotene in pigs is limited. Nevertheless beta-carotene might positively affect reproduction. In this study the absorption and tissue distribution of beta-carotene as well as its function as precursor of vitamin A was investigated in gilts that were fed according to one of three dietary treatments: VA (4000 IU vitamin A), VA + VA (4000 IU + 8300 IU) and VA + BC (4000 IU + 100 mg beta-carotene per kg diet) for 14 weeks. Only in the VA + BC group was beta-carotene detected in plasma (1-8 ng x mL(-1)), liver, adrenals and corpora lutea, indicating that pigs absorb intact beta-carotene at low rates. Liver levels of vitamin A were higher (P < 0.01) at comparable levels in the VA + VA and VA + BC group than in the VA group, indicating a conversion rate of beta-carotene to vitamin A of 40 to 1 on the basis of weight for beta-carotene at this level (100 mg x kg(-1)) in the diet. Higher levels of vitamin A in the uterus of the VA + BC group (P < 0.01) as well as the accumulation of beta-carotene in adrenals and corpora lutea might reflect some influence of beta-carotene on local vitamin A metabolism which might be of importance for reproductive performance in gilts.     

Comparative role of vitamin A and beta-carotene on reproduction and neonate survival in rats

Female rats were used to investigate the comparative role of vitamin A and beta-carotene (dietary or injected) on growth, feed intake and reproduction. After 3 wk of vitamin A and beta-carotene depletion, rats were assigned to one of six groups: 1) CON = fed 5% NRC recommended level of vitamin A (= 60 mug retinol equivalent (RE)/kg diet); 2) VA = fed 100% of vitamin A (= 1200 mug RE/kg diet); 3) HVA = fed 150% of vitamin A; 4) VA+C = fed 100% of vitamin A + 1.2mg beta-carotene; 5) VA+IC = fed 100% of vitamin A + injected weekly with 8.37 mg of beta-carotene; and 6) VA+IVA = fed 100% of vitamin A + injected weekly with 1400 IU of vitamin A. The level of vitamin A and beta-carotene in dam blood and liver reflected the level of supplementation. No difference in feed intake or body weight was observed. Although mean litter size was similar for all groups, mean pup weight at birth was lowest for deficient rats. Pup mortality through 2 wk postpartum was lower for groups receiving higher levels of either vitamin A or beta-carotene. However, supplemental beta-carotene did not influence growth or reproductive performance. Therefore, low intakes in vitamin A or beta-carotene had no effect on feed intake, growth or reproduction in female rats but decreased fetal growth and increased mortality among pups.     

Interrelationships between the gastric cytoprotective effects of vitamin A and beta-carotene and the gastric mucosal superoxide dismutase activity in rats

Gastric mucosal damage was produced in rats by the intragastric administration of 96% ethanol or 0.6 M HCl, according to the method of Robert et al. Vitamin A or beta-carotene, in doses of 10 mg/kg, given intragastrically 30 min before the administration of the necrotizing agents. The animals were killed 1 hr after the administration of the necrotizing agents. The following experimental parameters were studied, without and with application of vitamin A and beta-carotene; number of gastric lesions (ulcers); severity of gastric mucosal lesions (ulcers); gastric mucosal superoxide dismutase (SOD) activity. It was found that; vitamin A and beta-carotene, in doses of 10 mg/kg, are able to prevent significantly both the number and severity of gastric mucosal lesions (ulcers) produced by the application of 96% ethanol or 0.6 M HCl; the significant increase of ethanol-induced gastric mucosal SOD activity can be inhibited by the application of vitamin A and beta-carotene; vitamin A and beta-carotene are capable of preventing the development of gastric mucosal lesions (ulcers) produced by the intragastric administration of 0.6 M HCl, while these agents fail to compensate for the HCl-induced decrease of gastric mucosal SOD activity. It has been suggested that; vitamin A and beta-carotene are gastric cytoprotective agents; the ulcer preventive effects of vitamin A and beta-carotene are partly dependent on their scavanger behaviour.     

Absorption and retinol equivalence of beta-carotene in humans is influenced by dietary vitamin A intake

The effect of vitamin A supplements on metabolic behavior of an oral tracer dose of [14C]beta-carotene was investigated in a longitudinal test-retest design in two adults. For the test, each subject ingested 1 nmol of [14C]beta-carotene (100 nCi) in an emulsified olive oil-banana drink. Total urine and stool were collected for up to 30 days; concentration-time patterns of [14C]beta-carotene, [14C]retinyl esters, and [14C]retinol were determined for 46 days. On Day 53, the subjects were placed on a daily vitamin A supplement (10000 IU/day), and a second dose of [14C]beta-carotene (retest) was given on Day 74. All 14C determinations were made using accelerator mass spectrometry. In both subjects, the vitamin A supplementation was associated with three main effects: 1). increased apparent absorption: test versus retest values rose from 57% to 74% (Subject 1) and from 52% to 75% (Subject 2); 2). an approximately 10-fold reduction in urinary excretion; and 3). a lower ratio of labeled retinyl ester/beta-carotene concentrations in the absorptive phase. The molar vitamin A value of the dose for the test was 0.62 mol (Subject 1) and 0.54 mol (Subject 2) vitamin A to 1 mol beta-carotene. Respective values for the retest were 0.85 and 0.74. These results show that while less cleavage of beta-carotene occurred due to vitamin A supplementation, higher absorption resulted in larger molar vitamin A values.     

Photoprotective potential of lycopene,beta-carotene,vitamin E,vitamin C and carnosic acid in UVA-irradiated human skin fibroblasts

The photoprotective potential of the dietary antioxidants vitamin C, vitamin E, lycopene, beta-carotene, and the rosemary polyphenol, carnosic acid, was tested in human dermal fibroblasts exposed to ultraviolet-A (UVA) light. The carotenoids were prepared in special nanoparticle formulations together with vitamin C and/or vitamin E. Nanoparticle formulations, in contrast to dimethylsulphoxide, stablized lycopene in the cell culture medium and allowed efficient cellular uptake. The presence of vitamin E in the formulation further increased the stability and cellular uptake of lycopene. UVA irradiation of the human skin fibroblasts led to a 10-15-fold rise in metalloproteinase 1 (MMP-1) mRNA. This rise was suppressed in the presence of low microM concentrations of vitamin E, vitamin C, or carnosic acid but not with beta-carotene or lycopene. Indeed, in the presence of 0.5-1.0 microM beta-carotene or lycopene, the UVA-induced MMP-1 mRNA was further increased by 1.5-2-fold. This increase was totally suppressed when vitamin E was included in the nanoparticle formulation. Heme-oxygenase 1 (HO-1) mRNA expression was strongly induced by UVA irradiation but none of the antioxidants inhibited this effect at the concentrations used in this study. Indeed, beta-carotene or lycopene (0.5-1.0 microM) led to a further 1.5-fold rise in the UVA-induced HO-1 mRNA levels. In conclusion, vitamin C, vitamin E, and carnosic acid showed photoprotective potential. Lycopene and beta-carotene did not protect on their own but in the presence of vitamin E, their stability in culture was improved and the rise in MMP-1 mRNA expression was suppressed, suggesting a requirement for antioxidant protection of the carotenoids against formation of oxidative derivatives that can influence the cellular and molecular responses.     

Identification and characterization of a mammalian enzyme catalyzing the asymmetric oxidative cleavage of provitamin A

In vertebrates, symmetric versus asymmetric cleavage of beta-carotene in the biosynthesis of vitamin A and its derivatives has been controversially discussed. Recently we have been able to identify a cDNA encoding a metazoan beta,beta-carotene-15,15'-dioxygenase from the fruit fly Drosophila melanogaster. This enzyme catalyzes the key step in vitamin A biosynthesis, symmetrically cleaving beta-carotene to give two molecules of retinal. Mutations in the corresponding gene are known to lead to a blind, vitamin A-deficient phenotype. Orthologs of this enzyme have very recently been found also in vertebrates and molecularly characterized. Here we report the identification of a cDNA from mouse encoding a second type of carotene dioxygenase catalyzing exclusively the asymmetric oxidative cleavage of beta-carotene at the 9',10' double bond of beta-carotene and resulting in the formation of beta-apo-10'-carotenal and beta-ionone, a substance known as a floral scent from roses, for example. Besides beta-carotene, lycopene is also oxidatively cleaved by the enzyme. The deduced amino acid sequence shares significant sequence identity with the beta,beta-carotene-15,15'-dioxygenases, and the two enzyme types have several conserved motifs. To establish its occurrence in different vertebrates, we then attempted and succeeded in cloning cDNAs encoding this new type of carotene dioxygenase from human and zebrafish as well. As regards their possible role, the apocarotenals formed by this enzyme may be the precursors for the biosynthesis of retinoic acid or exert unknown physiological effects. Thus, in contrast to Drosophila, in vertebrates both symmetric and asymmetric cleavage pathways exist for carotenes, revealing a greater complexity of carotene metabolism.     

Dietary supplementation with antioxidants. Is there a case for exceeding the recommended dietary allowance?

The containment of damaging oxygen species by antioxidant nutrients has led to the speculation that the RDA for these specific nutrients may be overly low. Among these nutrients are vitamin E, vitamin C, and to a lesser extent beta-carotene and selenium. Evidence for the role of these nutrients in cancer and heart disease is evaluated. The case is presented for an increase of two-fold for the vitamin C RDA and between three and five-fold for vitamin E; for establishing 15 mg as the RDA for beta-carotene; for no change in the vitamin A RDA; and for further study on selenium.     

Vitamin E and beta-carotene status of dairy cows: a survey of plasma levels and supplementation practices

Culling rate in dairy cattle has increased considerably, thereby reducing cowś longevity and raising sustainability concerns worldwide. In the last decades, feeding systems have changed towards larger inclusion of preserved forages and reduced fresh herbage, which may limit vitamin E and beta-carotene dietary supply to dairy cows. Because of higher oxidative stress, engendered by greater milk production of modern genetics, the requirement for these nutrients is increased. Therefore, this study aimed to assess the current status of vitamin E and beta-carotene of commercial dairy cows. Blood vitamin E and beta-carotene concentrations were measured in 2 467 dairy cows from 127 farms in Belgium, Germany, Iberia and The Netherlands, that were visited once. Five cows were randomly selected per lactation stage per farm: Dry (between 30 and 1 day(s) before calving), Very-early (from calving until 15 days in milk (DIM)), Early (between 16 and 119 DIM), and Mid-late (from 120 DIM onwards). In addition, a survey was conducted to retrieve data on vitamin E and beta-carotene supplementation and feeding practices. Vitamin E and beta-carotene blood concentrations dropped considerably around calving. Among all surveyed cows, more than 75 and 44% were deficient in vitamin E and beta-carotene (i.e., blood concentration below 3.0 and 3.5 mg/l, respectively). Of the Very-early group, more than 97 and 78% of the cows were deficient in vitamin E and beta-carotene, respectively, with respective blood concentrations of 1.15 and 2.71 mg/l, which was significantly lower than the other lactation stages. Vitamin E and beta-carotene blood concentrations, as well as their supplementation levels, significantly varied among countries. Vitamin E and beta-carotene blood concentrations were positively related to the total estimated daily intakes of vitamin E and beta-carotene. Therefore, blood concentrations of vitamin E and beta-carotene depend on their respective level of intake, which is generally below recommendations and varies greatly between countries. Supplementation could contribute to provide cows with adequate amounts of vitamin E and beta-carotene all along the lactation, to ensure their lifetime performance and improve their fertility.     

Inhibition of immunoglobulin E production in allergic model mice by supplementation with vitamin E and beta-carotene

A diet containing different amounts of vitamin E (alpha-tocopherol; 0.5 mg, 5 mg, 10 mg or 50 mg per 100 g diet) was supplemented to BALB/c mice for 6 weeks. These mice were subcutaneously immunized twice with ovalbumin (OVA). A passive cutaneous anaphylaxis (PCA) analysis demonstrated that the mice fed on the diet containing 5 mg of vitamin E produced the highest level of the OVA-specific immunoglobulin E (IgE) antibody. A lower level of serum IgE was found in the mice supplemented with 0.5 mg, 10 mg and 50 mg of vitamin E. A sandwich ELISA analysis showed that the pattern of the total IgE antibody level among these four groups was the same as that of the allergen-specific IgE. In a separate experiment, 5 mg of vitamin E and/or 50 mg of beta-carotene was supplemented to the basal diet containing vitamin E as alpha-tocopherol acetate (5 mg) in order to evaluate the effect of their combination on OVA-specific and total IgE production in the mice. The supplementation with beta-carotene alone had no effect on OVA-specific or total IgE production. In contrast, supplementation with vitamin E plus beta-carotene effectively suppressed both the antigen-specific and total IgE antibodies. The serum vitamin E and beta-carotene levels were increased by supplementation with the respective compounds. These results strongly suggest that the combination of dietary vitamin E and beta-carotene suppressed IgE production and would therefore help to prevent the type-I allergic reaction.     

Peroxidized cholesterol-induced matrix metalloproteinase-9 activation and its suppression by dietary beta-carotene in photoaging of hairless mouse skin

The activation of matrix metalloproteinase (MMP)-9 leading to the formation of wrinkle and sagging of skin is an essential step in the skin photoaging on exposure to ultraviolet A (UVA). This study attempted to elucidate the role of peroxidized cholesterol including cholesterol hydroperoxides (Chol-OOHs), primary products of lipid peroxidation in biomembranes, in MMP-9 activation and the effect of dietary beta-carotene in MMP-9 activation. Hairless mice were subjected to periodic UVA irradiation for 8 weeks. The amount of peroxidized cholesterol detected as total hydroxycholesterol in the skin was increased significantly by the exposure. The activity and protein level of MMP-9 were elevated with wrinkling and sagging formation. MMP-9 activity was also enhanced by the intracutaneous injection of Chol-OOHs into the mouse skin. Adding beta-carotene to the diet of the mice during the period of irradiation suppressed the activity and expression of MMP-9 as well as the wrinkling and sagging formation. The amount of cholesterol 5alpha-hydroperoxide, a singlet molecular oxygen oxygenation-specific peroxidized cholesterol, was significantly lowered by the addition of beta-carotene to the diet. These results strongly suggest that Chol-OOHs formed on exposure to UVA contribute to the expression of MMP-9, resulting in photoaging. Dietary beta-carotene prevents the expression of MMP-9, at least partly, by inhibiting photodynamic action involved in the formation of Chol-OOHs.     

Metabolism and biological potency of 5,6-monoepoxy-β-carotene and 5,6:5′,6′-diepoxy-β-carotene

1. 5,6-Monoepoxy-beta-carotene and 5,6:5',6'-diepoxy-beta-carotene were partially converted into the furanoid forms during passage through the rat stomach. 2. The monoepoxide was converted into vitamin A in the small intestine and showed a biological potency 21% of that of beta-carotene. Neither beta-carotene nor 5,6-monoepoxyvitamin A was formed. 3. Intraperitoneal administration of the monoepoxide led to the accumulation of the unchanged compound in the liver and other tissues. 4. The diepoxide gave no beta-carotene or vitamin A or 5,6-monoepoxyvitamin A when given orally and showed no biological potency. 5. The significance of these results with special reference to the mechanism of formation of vitamin A from beta-carotene is discussed.     

Carotene cleavage activity in bovine ovarian follicles

Carotene cleavage activity has been demonstrated in bovine ovarian follicles by incubating cell-free homogenates from granulosa cells with 15, 15'-(14)C-beta-carotene. Enzyme activity was highly dependent on the quality of follicles studied. Highest cleavage activity was found in large, preovulatory follicles. The correlation between follicular fluid concentration of vitamin A and the conversion rate obtained with the corresponding enzyme preparations was significantly positive. No correlation, however, could be shown between cleavage activity and beta-carotene, alpha-tocopherol or cholesterol. The only form of vitamin A in follicular fluid of cattle was found to be retinol. The results support the hypothesis that a local conversion of beta-carotene into vitamin A in follicular structures is responsible for the increase of the intrafollicular concentration of vitamin A during follicular development.     

Cell type-specific expression of beta-carotene 9',10'-monooxygenase in human tissues.

The symmetrically cleaving beta-carotene 15,15'-monooxygenase (BCO1) catalyzes the first step in the conversion of provitamin A carotenoids to vitamin A in the mucosa of the small intestine. This enzyme is also expressed in epithelia in a variety of extraintestinal tissues. The newly discovered beta-carotene 9',10'-monooxygenase (BCO2) catalyzes asymmetric cleavage of carotenoids. To gain some insight into the physiological role of BCO2, we determined the expression pattern of BCO2 mRNA and protein in human tissues. By immunohistochemical analysis it was revealed that BCO2 was detected in cell types that are known to express BCO1, such as epithelial cells in the mucosa of small intestine and stomach, parenchymal cells in liver, Leydig and Sertoli cells in testis, kidney tubules, adrenal gland, exocrine pancreas, and retinal pigment epithelium and ciliary body pigment epithelia in the eye. BCO2 was uniquely detected in cardiac and skeletal muscle cells, prostate and endometrial connective tissue, and endocrine pancreas. The finding that the BCO2 enzyme was expressed in some tissues and cell types that are not sensitive to vitamin A deficiency and where no BCO1 has been detected suggests that BCO2 may also be involved in biological processes other than vitamin A synthesis.     

Dissection of the pathway required for generation of vitamin A and for Drosophila phototransduction

Dietary carotenoids are precursors for the production of retinoids, which participate in many essential processes, including the formation of the photopigment rhodopsin. Despite the importance of conversion of carotenoids to vitamin A (all-trans-retinol), many questions remain concerning the mechanisms that promote this process, including the uptake of carotenoids. We use the Drosophila visual system as a genetic model to study retinoid formation from beta-carotene. In a screen for mutations that affect the biosynthesis of rhodopsin, we identified a class B scavenger receptor, SANTA MARIA. We demonstrate that SANTA MARIA functions upstream of vitamin A formation in neurons and glia, which are outside of the retina. The protein is coexpressed and functionally coupled with the beta, beta-carotene-15, 15'-monooxygenase, NINAB, which converts beta-carotene to all-trans-retinal. Another class B scavenger receptor, NINAD, functions upstream of SANTA MARIA in the uptake of carotenoids, enabling us to propose a pathway involving multiple extraretinal cell types and proteins essential for the formation of rhodopsin.     

Vitamin A deficiency reduces uptake of beta-carotene by brush border membrane vesicles but does not alter intestinal retinyl ester hydrolase activity in the rat

Vitamin A deficiency has been reported to result in mild structural and functional changes within the small intestine. The objective of this study was to measure the impact of vitamin A deficiency in the rat on several functional aspects of beta-carotene uptake and intestinal retinyl ester hydrolysis. These included uptake of (14)C-beta-carotene by brush border membrane vesicles (BBMV) and in vitro activity of intrinsic retinyl ester hydrolase (REH). Rats (n = 33) were randomly assigned to receive one of three dietary treatments: vitamin A deficient (-VA), vitamin A sufficient pair-fed (PF), or vitamin A sufficient free access-fed (FA). Liver, serum retinol, and growth data were used to verify clinical vitamin A deficiency. Rats in the -VA group were clinically vitamin A deficient by Day 56 on a vitamin A-free diet and, at that point, all rats were randomly assigned to one of two experimental treatments: BBMV studies or REH activity assays. Uptake of (14)C-beta-carotene by BBMV was significantly suppressed (P < 0.05) in -VA rats when compared to both PF and FA control rats during early passive uptake equilibration (10-20 sec). Uptake was also significantly decreased by BBMV isolated from -VA rats compared to PF controls, but not FA controls, after a 10-min incubation (P < 0.05). In vitro activity of REH was not impacted by vitamin A deficiency in rats, although a trend for greater activity from -VA rats was noted. These data suggest that vitamin A deficiency impairs enterocyte membrane uptake of beta-carotene without altering the enzymatic activity of intrinsic REH.     

Beta-carotene from cassava (Manihot esculenta Crantz) leaves improves vitamin A status in rats

The bioavailability of beta-carotene from cassava (Manihot esculenta Crantz) leaves was assayed in vitamin A deficient Wistar rats (Rattus norvegicus). Rats were separated into three groups and fed with a modified AIN-93G--vitamin A deficient--diet. Deficient rat received this diet without any additional vitamin A source. Controls received the diet with 7200 microg of synthetic beta-carotene (control), while experimentals (test) received 19.5 g of cassava leaves powder per kg of diet. The cassava leaves with beta-carotene promotes similar growth and tissue weight in rats to the synthetic beta-carotene. The relative bioavailability, estimated as the Retinol Accumulation Factor (RAF), was 16.5 and 27.5 for control and test groups, respectively, indicating that control and test rats should have an intake of 16.5 microg or 27.5 microg of beta-carotene from synthetic form or cassava leaves powder for each 1 microg of hepatic retinol stored, respectively. The cassava leaves beta-carotene bioavailability was lower than the synthetic beta-carotene probably because the beta-carotene from the leaf matrix may be bounded to protein complex or inside organelles, which impair carotenoid absorption. Our findings showed that beside the hepatic retinol recovery, cassava leaf beta-carotene could maintain rat growth and avoid vitamin A deficient symptoms.     

Enhancement of the UVA induction of haem oxygenase-1 expression by beta-carotene in human skin fibroblasts.

beta-Carotene has often been discussed as a means to reduce the risk of skin photodamage. We studied the antioxidative potential of beta-carotene in human skin fibroblasts exposed to ultraviolet A light. Surprisingly, we found a pro-oxidative effect of beta-carotene. Using the induction of haem oxygenase-1 as a marker for oxidative stress, we found a strong enhancement of gene expression by beta-carotene in ultraviolet A-irradiated cells. This effect was clearly suppressed by concomitant addition of vitamin E but only moderately by vitamin C. The results show that beta-carotene has pro-oxidative properties in human skin fibroblasts exposed to ultraviolet-A light.     

Carotenoid and retinoid metabolism: insights from isotope studies

Use of isotopes as tracers has had an important role in elucidating key features of vitamin A and retinoid metabolism in animal models and humans. Their use has shown that beta-carotene absorption is variable, and that the appearance of beta-carotene and its metabolites in the blood by time since dosing follows characteristic patterns. Retinol formed from beta-carotene shows a different pattern, as does lutein. In this article, we summarize and discuss insights and some surprises into the absorption and metabolism of vitamin A, beta-carotene, and lutein that were gained with the use of isotope tracers in humans, rats, and cells as models.     

Vitamin A Intake and Risk of Melanoma: A Meta-Analysis

Background

Mounting evidence from experimental and animal studies suggests that vitamin A may have a protective effect on melanoma, but the findings on the association of vitamin A intake with risk of melanoma have been inconsistently reported in epidemiologic studies. We attempted to elucidate the association by performing a meta-analysis.

Methods

Eligible studies were identified by searching PubMed and EMBASE databases, as well as by reviewing the references of retrieved publications. Summary odds ratios (OR) with corresponding 95% confidence interval (CI) were computed with a random-effects model. Study-specific ORs and 95% CIs for the highest vs. lowest categories of vitamin A intake were pooled.

Results

A total of 8 case-control studies and 2 prospective studies comprising 3,328 melanoma cases and 233,295 non-case subjects were included. The summary OR for the highest compared with the lowest intake of total vitamin A, retinol and beta-carotene was 0.86 (95% CI = 0.59–1.25), 0.80 (95% CI = 0.69–0.92) and 0.87 (95%CI = 0.62–1.20), respectively. Significant heterogeneity was observed among studies on vitamin A and beta-carotene intake, but not among studies on retinol intake. Subgroup and sensitivity analyses confirmed these findings. There was no indication of publication bias.

Conclusion

Findings from this meta-analysis suggest that intake of retinol, rather than of total vitamin A or beta-carotene, is significantly associated with reduced risk of melanoma.