Use of Theoretical Subjects to Develop a Method for Assessing Equivalence of Dietary Vitamin A in a Mixed Diet.

BACKGROUND: Although the vitamin A (VA) equivalency of provitamin A carotenoids from single foods or capsules has been studied using several approaches, there is currently no reliable method to determine VA equivalency for mixed diets. OBJECTIVES: To reach the objective of identifying a method to determine the VA equivalency of provitamin A carotenoids in mixed diets, we tested a new approach using preformed VA as proxy for provitamin A. METHODS: We studied 6 theoretical subjects who were assigned physiologically plausible values for dietary VA intake, retinol kinetic parameters, plasma retinol pool size, and VA total body stores. Using features in the Simulation, Analysis and Modeling software, we specified that subjects ingested a tracer dose of stable isotope-labeled VA on day 0 followed by 0-µg supplemental VA or 200, 400, 800, 1200, 1600, and 2000 µg VA daily from day 14 to day 28; we assigned VA absorption to be 75%. For each supplement level, we simulated plasma retinol specific activity (SAp) over time and calculated the mean decrease in SAp relative to 0 µg. Group mean data were fitted to a regression equation to calculate predicted VA equivalency at each supplement level on day 28. RESULTS: For each subject, higher VA supplement loads resulted in lower SAp, with the magnitude of the decrease differing among subjects. The mean predicted amount of absorbed VA was within 25% of individual subjects' assigned amount for 4 of the 6 subjects, and the mean ratio of predicted to assigned amount of absorbed VA over all supplement loads ranged from 0.60 to 1.50, with an overall mean ratio of 1.0. CONCLUSIONS: Results for preformed VA suggest that this protocol may be useful for determining VA equivalency of provitamin A carotenoids in free-living subjects if mixed diets with known provitamin A content were substituted for the VA supplements.

PLASMA VITAMIN AND MINERAL CONCENTRATIONS IN CAPTIVE GREATER FLAMINGOS (PHOENICOPTERUS ROSEUS) AS INFLUENCED BY DIET CHANGE.

The greater flamingo (Phoenicopterus roseus) is often maintained in managed care. Although values for plasma concentrations of minerals and vitamins have been published for this species, limited studies investigate the effects of diet changes on these values. Plasma concentrations of selected vitamins (A [and the provitamin A carotenoid ß-carotene] and E), minerals (Ca, Cu, Fe, K, Mg, Mn, Na, inorganic P, Se, and Zn), and total protein (TP) were measured in a zoo collection of greater flamingos in November 2018 (n = 48) and in November 2019 (n = 52), 30 d after a change from the summer diet, which was the same in both years, to different winter diets. Chloride, cholesterol, and triglycerides were only measured once in 2019. The nutrient profiles of the two complete diets (labeled and analyzed) differed considerably, especially for the canthaxanthin concentration. The plasma concentrations of ß-carotene (P < 0.0001), Cu (P < 0.0001), Fe (P < 0.0001), Mg (P < 0.0001), and Se (P < 0.0001) were significantly higher and vitamin E (P < 0.0001), Ca (P = 0.0014), Mn (P < 0.0001), Na (P = 0.0005), and TP (P < 0.0001) were significantly lower in 2018 than in 2019. Four minerals (Cu, Fe, Mg, and Zn) were measured using both spectrophotometry and inductively coupled plasma mass spectrometry, and all of these minerals showed significant (P < 0.05) differences between the two methods. Based on the results of the study, both the influence of diet and the detection method used for circulating nutrients must be considered when interpreting the plasma vitamin and mineral concentrations in greater flamingos.

Changes in carbon allocation and subplastidal amyloplast structures of specialised Ipomoea batatas (sweet potato) storage root phenotypes.

Vitamin A deficiency (VAD) in Low and Medium Income countries remains a major health concern. Ipomoea batatas, orange sweet potato (OSP), is one of the biofortification solutions being implemented by the World Health Organisation (WHO) to combat VAD. However, high provitamin A (ß-carotene) content has been associated with a reduction in dry matter, reducing calorific value and having adverse effects on consumer traits. Both starch and carotenoid formation are located in amyloplasts and could potentially compete for the same precursors. Hence, five different sweet potato storage root phenotypes were characterized through spatial metabolomics and proteomics at the sub-plastidal level. The metabolite data suggested an indirect correlation of starch and carotenoids through the TCA cycle and pentose phosphate pathway. Furthermore, a change in lipid composition was observed to accommodate the storage of carotenoids in the hydrophilic environment of the amyloplast. The data suggests an alteration of cellular ultra-structures and perturbation of metabolism in high ß-carotene producing sweet potato roots. This corroborates with previous gene expression analysis through biochemical analysis of sweet potato root tissue.

Use of stable isotopes to study bioconversion and bioefficacy of provitamin A carotenoids.

Provitamin A carotenoids (pVACs) are important contributors to vitamin A status and in reducing vitamin A deficiency in vulnerable populations. However, there are uncertainties about the effectiveness of dietary pVACs to provide sufficient amounts of vitamin A due to large variations in provitamin A bioefficacy, and if provitamin A carotenoids other than ß-carotene could be utilized in biofortification programs. Although animal studies have compared the bioefficacy of ß-cryptoxanthin and ß-carotene in biofortified maize by measuring liver retinol concentrations after dietary exposure, it is unclear whether the higher bioavailability of ß-cryptoxanthin can be offset by the lower conversion of ß-cryptoxanthin to retinol, and how post-intestinal conversion of ß-cryptoxanthin may contribute to the total bioefficacy of ß-cryptoxanthin. Here, we present a method that, for the first time, uses stable isotope labeled ß-cryptoxanthin and ß-carotene to quantify bioconversion and bioefficacy of both pVACs simultaneously in human volunteers. The paper describes how positioning of the [13C] labels around the centric 15,15' double bond on either the ß-carotene or ß-cryptoxanthin molecule allows quantification of retinoids from both pVACs, and details the procedure for sample preparation and analysis using LC-MS/MS. Finally, we apply and discuss recent approaches to quantify bioconversion and bioefficacy of isotopically labeled ß-carotene and ß-cryptoxanthin in humans.

Vitamin A5/X, a New Food to Lipid Hormone Concept for a Nutritional Ligand to Control RXR-Mediated Signaling.

Vitamin A is a family of derivatives synthesized from carotenoids acquired from the diet and can be converted in animals to bioactive forms essential for life. Vitamin A1 (all-trans-retinol/ATROL) and provitamin A1 (all-trans-ß,ß-carotene/ATBC) are precursors of all-trans-retinoic acid acting as a ligand for the retinoic acid receptors. The contribution of ATROL and ATBC to formation of 9-cis-13,14-dihydroretinoic acid (9CDHRA), the only endogenous retinoid acting as retinoid X receptor (RXR) ligand, remains unknown. To address this point novel and already known retinoids and carotenoids were stereoselectively synthesized and administered in vitro to oligodendrocyte cell culture and supplemented in vivo (orally) to mice with a following high-performance liquid chromatography-mass spectrometry (HPLC-MS)/UV-Vis based metabolic profiling. In this study, we show that ATROL and ATBC are at best only weak and non-selective precursors of 9CDHRA. Instead, we identify 9-cis-13,14-dihydroretinol (9CDHROL) and 9-cis-13,14-dihydro-ß,ß-carotene (9CDHBC) as novel direct nutritional precursors of 9CDHRA, which are present endogenously in humans and the human food chain matrix. Furthermore, 9CDHROL displayed RXR-dependent promnemonic activity in working memory test similar to that reported for 9CDHRA. We also propose that the endogenous carotenoid 9-cis-ß,ß-carotene (9CBC) can act as weak, indirect precursor of 9CDHRA via hydrogenation to 9CDHBC and further metabolism to 9CDHROL and/or 9CDHRA. In summary, since classical vitamin A1 is not an efficient 9CDHRA precursor, we conclude that this group of molecules constitutes a new class of vitamin or a new independent member of the vitamin A family, named "Vitamin A5/X".

Development of sub-tropically adapted diverse provitamin-A rich maize inbreds through marker-assisted pedigree selection, their characterization and utilization in hybrid breeding.

Malnutrition has emerged as one of the major health problems worldwide. Traditional yellow maize has low provitamin-A (proA) content and its genetic base in proA biofortification breeding program of subtropics is extremely narrow. To diversify the proA rich germplasm, 10 elite low proA inbreds were crossed with a proA rich donor (HP702-22) having mutant crtRB1 gene. The F2 populations derived from these crosses were genotyped using InDel marker specific to crtRB1. Severe marker segregation distortion was observed. Seventeen crtRB1 inbreds developed through marker-assisted pedigree breeding and seven inbreds generated using marker-assisted backcross breeding were characterized using 77 SSRs. Wide variation in gene diversity (0.08 to 0.79) and dissimilarity coefficient (0.28 to 0.84) was observed. The inbreds were grouped into three major clusters depicting the existing genetic diversity. The crtRB1-based inbreds possessed high ß-carotene (BC: 8.72µg/g), ß-cryptoxanthin (BCX: 4.58µg/g) and proA (11.01µg/g), while it was 2.35µg/g, 1.24µg/g and 2.97µg/g in checks, respectively. Based on their genetic relationships, 15 newly developed crtRB1-based inbreds were crossed with five testers (having crtRB1 gene) using line × tester mating design. 75 experimental hybrids with crtRB1 gene were evaluated over three locations. These experimental hybrids possessed higher BC (8.02µg/g), BCX (4.69µg/g), proA (10.37µg/g) compared to traditional hybrids used as check (BC: 2.36 µg/g, BCX: 1.53µg/g, proA: 3.13µg/g). Environment and genotypes × environment interaction had minor effects on proA content. Both additive and dominance gene action were significant for proA. The mean proportion of proA to total carotenoids (TC) was 44% among crtRB1-based hybrids, while 11% in traditional hybrids. BC was found to be positively correlated with BCX (r = 0.68) and proA (r = 0.98). However, no correlation was observed between proA and grain yield. Several hybrids with >10.0 t/ha grain yield with proA content >10.0 µg/g were identified. This is the first comprehensive study on development of diverse proA rich maize hybrids through marker-assisted pedigree breeding approach. The findings provides sustainable and cost-effective solution to alleviate vitamin-A deficiency.

Temperature and storage time increase provitamin A carotenoid concentrations and bioaccessibility in post-harvest carrots.

The aim was to enhance provitamin A carotenoid (proVA CAR) concentrations and bioaccessibility in carrots by manipulating post-harvest factors. To that end, we assessed the effects of Ultraviolet-C light, pulsed light, storage temperature, and storage duration. We also measured CAR bioaccessibility by using an in vitro model. Pulsed light, but not Ultraviolet-C, treatment increased proVA CAR concentrations in the cortex tissue (p < 0.05). Longer storage times and higher temperatures also increased concentrations (p < 0.05). The maximal increase induced by pulsed light was obtained after treatment with 20 kJ/m2 and 3-days of storage at 20 °C. However, the positive effect induced by pulsed light decreased considerably over the next seven days. ProVA CAR in carrots with the highest concentrations also proved to be more bioaccessible (p < 0.05). Thus, proVA CAR concentrations in stored carrots can be increased significantly through storage times and temperatures. Pulsed light can also significantly increase proVA CAR concentrations, but only temporarily.

Genetic diversity among early provitamin A quality protein maize inbred lines and the performance of derived hybrids under contrasting nitrogen environments.

BACKGROUND: Information on population structure and genetic diversity of germplasm in a breeding programme is useful because it enhances judicious utilisation of genetic resources to achieve breeding objectives. Seventy early maturing provitamin A (PVA) quality protein maize (QPM) inbreds developed by the IITA- maize improvement programme were genotyped using 8171 DArTseq markers. Furthermore, 96 hybrids derived from 24 selected inbreds plus four checks were evaluated under low-N and optimal environments in Nigeria during 2016 and 2017. Genotypic and phenotypic data of inbreds and hybrids respectively, were analysed to (i) assess the level of genetic dissimilarities and population structure of the inbreds, and (ii) investigate the grain yield performance of derived hybrids under low-N, optimal and across environments. RESULTS: Genetic diversity among the seventy inbreds was high varying from 0.042 to 0.500 with an average of 0.357. Sixty-six inbred lines with probabilities ≥0.70 were assigned to a single group. The population structure analysis, the UPGMA phylogeny, and the principal Coordinate Analysis (PCoA) of the DArTseq markers revealed a clear separation of five groups and each followed pedigree records. Clustered inbreds displayed common characteristics including high PVA levels, and drought and low-N tolerance. The top performing hybrid, TZEIORQ 40 × TZEIORQ 26 out-yielded the best hybrid control, TZEIOR 127 × TZEIOR 57 by 8, 3, and 9% under low-N, optimal, and across environments, respectively. High repeatability estimates were detected for grain yield under each and across environments. Similarly, high breeding efficiency of 71, 70 and 72% were computed under low-N, optimal, and across environments, respectively. CONCLUSIONS: The UPGMA clustering, the structure analysis, and the PCoA consistently revealed five groups which largely followed pedigree information indicating the existence of genetically distinct groups in the inbred lines. High repeatability and breeding efficiency values estimated for grain yield of hybrids under low-N, optimal and across environments demonstrated that high productive hybrids could be developed using inbreds from the opposing clusters identified by the DArTseq markers. The 15 top performing hybrids identified, particularly TZEIORQ 40 × TZEIORQ 26 and TZEIORQ 29 × TZEIORQ 43 should be further evaluated for release and commercialization in SSA.

Molecular characterization and safety assessment of biofortified provitamin A rice.

Part of the studies involved in safety assessment of genetically engineered crops includes characterizing the organization, integrity, and stability of the inserted DNA and evaluating the potential allergenicity and toxicity of newly-expressed proteins. Molecular characterization of the introduced DNA in provitamin A biofortified rice event GR2E confirmed insertion of a single copy of the transfer-DNA in the genome and its inheritance as a single locus. Nucleotide sequencing of the inserted DNA confirmed it was introduced without modifications. The phytoene synthase, and carotene desaturase proteins did not display sequence similarity with allergens or toxins. Both proteins were rapidly digested in simulated gastric fluid and their enzymatic activity was inhibited upon heat treatment. Acute oral toxicity testing of the protein in mice demonstrated lack of adverse effects. These evidences substantiated the lack of any identifiable hazards for both proteins and in combination with other existing comparative analyses provided assurance that food derived from this rice is safe. This conclusion is in line with those of the regulatory agencies of US Food and Drug Administration, Health Canada and Food Standard Australia and New Zealand.

Pro-vitamin A carotenoid content of 48 plantain (Musa AAB genome) cultivars sourced from eastern Democratic Republic of Congo.

BACKGROUND: Vitamin A deficiency (VAD) is widespread in sub-Saharan Africa (SSA). Unlike in developed countries, where the main source of vitamin A comes from meat, the diet of poor populations in SSA is largely plant based. It is thus important to identify local / popular plants with higher vitamin A content for combating VAD. Banana (including plantains) is an important staple food crop in this region. The identification and promotion of vitamin A-rich banana cultivars could contribute significantly to the alleviation of VAD in areas heavily dependent on the crop. We assessed pro-vitamin A carotenoid (pVACs) content in the fruit pulp of 48 local plantains from eastern Democratic Republic of Congo, to identify cultivars that could help reduce VAD, especially among young children and women of reproductive age. RESULTS: Mean pVACs content varied from 175-1756 µg/100 gfw in ripe fruits. Significant increases (P < 0.001) in total pVACs content occurred after ripening in all cultivars except 'UCG II'. Retinol activity equivalents (RAE) in ripe fruits ranged from 12-113 µg/100 gfw. Fifteen plantain cultivars, including 'Adili II', 'Nzirabahima', 'Mayayi', 'Buembe', and 'Sanza Tatu' (associated with RAE values of 44 µg/100 gfw and above) can be considered as good sources of pVACs. Modest consumption (250 or 500 gfw) of the fruit pulp of the five best plantain cultivars at ripening stage 5 meets between 39-71% and 44-81% of vitamin A dietary reference intake (DRI) respectively, for children below 5 years old and women of reproductive age. CONCLUSION: The 15 best plantain cultivars (especially the top 5) could potentially be introduced / promoted as alternative sources of pro-vitamin A in banana-dependent communities, and help to reduce cases of VAD substantially. © 2019 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Determination of carotenoids in sweet potato (Ipomoea batatas L., Lam) tubers: Implications for accurate provitamin A determination in staple sturdy tuber crops.

Vitamin A deficiency (VAD) is a global health problem, which despite significant financial investments and initiatives has not been eradicated. Biofortification of staple crops with ß-carotene (provitamin A) in Low Medium Income Countries (LMICs) is the approach advocated and adopted by the WHO and HarvestPlus programme. The accurate determination of ß-carotene is key to the assessment of outputs from these activities. In the present study, HPLC-PDA analysis displayed superior resolving power, separating and identifying 23 carotenoids in the orange sweet potato (Ipomoea batatas) variety used, including only eight carotenoids with provitamin A properties. Additionally, the results evidently displayed that the use of lyophilised material facilitated the extraction of twice the amount of pigments compared to fresh material, which impacts the precise calculation of the provitamin A content. These results highlight that yellow to orange starchy edible crops produce a wide array of carotenoids in addition to ß-carotene. Biosynthetically it is clear from the intermediates and products accumulating that the ß-branch of the carotenoid pathway persists in sweet potato tuber material. Collectively, the data also have implications with respect to the determination and biosynthesis of provitamin A among staple crops for developing countries.

Compositional Analysis of Genetically Engineered GR2E "Golden Rice" in Comparison to That of Conventional Rice.

Compositional analyses were performed on samples of rice grain, straw, and derived bran obtained from golden rice event GR2E and near-isogenic control PSBRc82 rice grown at four locations in the Philippines during 2015 and 2016. Grain samples were analyzed for key nutritional components, including proximates, fiber, polysaccharides, fatty acids, amino acids, minerals, vitamins, and antinutrients. Samples of straw and bran were analyzed for proximates and minerals. The only biologically meaningful difference between GR2E and control rice was in levels of ß-carotene and other provitamin A carotenoids in the grain. Except for ß-carotene and related carotenoids, the compositional parameters of GR2E rice were within the range of natural variability of those components in conventional rice varieties with a history of safe consumption. Mean provitamin A concentrations in milled rice of GR2E can contribute up to 89-113% and 57-99% of the estimated average requirement for vitamin A for preschool children in Bangladesh and the Philippines, respectively.

Nutritional Properties and Consumer's Acceptance of Provitamin A-Biofortified Amahewu Combined with Bambara (Vigna Subterranea) Flour.

Amahewu is a fermented non-alcoholic cereal grain beverage, popular in Southern Africa. This study evaluates the possibility of producing an acceptable provitamin A (PVA)-biofortified maize amahewu, complemented with bambara flour, to contribute towards the alleviation of protein energy malnutrition (PEM) and vitamin A deficiency (VAD). Germinated, roasted, and raw bambara flours, were added at 30% (w/w) substitution level, separately, to either white maize or PVA-biofortified maize flour, and processed into amahewu. Wheat bran (5% w/w) was used as reference inoculum. Amahewu samples were analyzed for nutritional properties and acceptability. The protein and lysine contents of amahewu almost doubled with the inclusion of germinated bambara. Protein digestibility of amahewu samples increased by almost 45% with the inclusion of bambara. PVA-biofortified maize amahewu samples complemented with bambara were extremely liked for their color, aroma, and taste when compared with their white maize counterparts. The principal component analysis explained 96% of the variation and PVA-biofortified maize amahewu samples were differentiated from white maize amahewu samples. The taste of amahewu resulting from roasting and germination of bambara was preferred in PVA-biofortified maize amahewu, compared to white maize amahewu. We conclude that PVA-biofortified maize amahewu, complemented with germinated bambara, has the potential to contribute towards the alleviation of PEM and VAD.

Carotenoids retention in biofortified yellow cassava processed with traditional African methods.

BACKGROUND: Biofortified yellow cassava is being cultivated in countries with high cassava consumption to improve its population's vitamin A status. The carotenoid retention in biofortified cassava when processed as boiled, fufu, and chikwangue was evaluated in this study. Commercial biofortified varieties Kindisa and Vuvu and the experimental genotypes MVZ2011B/360 and MVZ2012/044 were used. Fresh cassava roots were processed as boiled, fufu, and chikwangue. Provitamin A carotenoids (pVACs) content of fresh and processed cassava was measured by high-performance liquid chromatography, and total carotenoids was measured by spectrophotometer. RESULTS: pVACs content of fresh peeled cassava was 1.79-6.65 µg g-1 on a fresh weight basis, whereas in boiled cassava, fufu, and chikwangue the pVACs content was 1.71-6.91 µg g-1 , 0.04-0.37 µg g-1 , and 0.52-1.75 µg g-1 respectively. True retention of carotenoids after cooking was 93.2-96.8%, 0.8-3.1%, and 4.0-18.1% for boiled cassava, fufu, and chikwangue respectively. Significant total carotenoids loss was observed during storage. CONCLUSION: The results indicated that biofortified boiled cassava could be an effective food product to improve pVACs intake in areas where vitamin A deficiency exists, and processing of chikwangue and fufu should be improved before promoting biofortified cassava in vitamin-A-deficient areas with high cassava consumption. © 2018 The Authors. Journal of the Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Genetic variation of carotenoids, vitamin E and phenolic compounds in Provitamin A biofortified maize.

BACKGROUND: Biofortified maize is not only a good vehicle for provitamin A carotenoids for vitamin A deficient populations in developing countries but also a source of vitamin E, tocochromanols and phenolic compounds, which have antioxidant properties. Using high-performance liquid chromatography and a total antioxidant performance assay, the present study analyzed the antioxidant variation and antioxidant activity of 36 provitamin A improved maize hybrids and one common yellow maize hybrid. RESULTS: The ranges of major carotenoids in provitamin A carotenoids biofortified maize were zeaxanthin [1.2-13.2 µg g-1 dry weight (DW)], ß-cryptoxanthin (1.3-8.8 µg g-1 DW) and ß-carotene (1.3-8.0 µg g-1 DW). The ranges of vitamin E compounds identified in provitamin A carotenoids biofortified maize were α-tocopherol (3.4-34.3 µg g-1 DW), γ-tocopherol (5.9-54.4 µg g-1 DW), α-tocotrienol (2.6-19.5 µg g-1 DW) and γ-tocotrienol (45.4 µg g-1 DW). The ranges of phenolic compounds were γ-oryzanol (0.0-0.8 mg g-1 DW), ferulic acid (0.4-3.6 mg g-1 DW) and p-coumaric acid (0.1-0.45 mg g-1 DW). There was significant correlation between α-tocopherol and cis isomers of ß-carotene (P < 0.01). Tocotrienols were correlated with α-tocopherol and γ-oryzanol (P < 0.01). CONCLUSION: Genotype was significant in determining the variation in ß-cryptoxanthin, ß-carotene, α-tocopherol and γ-tocopherol contents (P < 0.01). A genotype × environment interaction was observed for γ-tocopherol content (P < 0.01). © 2016 Society of Chemical Industry.

Effect of thermal processing on the profile of bioactive compounds and antioxidant capacity of fermented orange juice.

Previously, we reported that alcoholic fermentation enhanced flavanones and carotenoids content of orange juice. The aim of this work was to evaluate the influence of pasteurization on the qualitative and quantitative profile of bioactive compounds and the antioxidant capacity of fermented orange juice. Ascorbic acid (203 mg/L), total flavanones (647 mg/L), total carotenoids (7.07 mg/L) and provitamin A (90.06 RAEs/L) values of pasteurized orange beverage were lower than those of fermented juice. Total phenolic remained unchanged (585 mg/L) and was similar to that of original juice. The flavanones naringenin-7-O-glucoside, naringenin-7-O-rutinoside, hesperetin-7-O-rutinoside, hesperetin-7-O-glucoside and isosakuranetin-7-O-rutinoside, and the carotenoids karpoxanthin and isomer, neochrome, lutein, ζ-carotene, zeaxanthin, mutatoxanthin epimers, ß-cryptoxanthin and auroxanthin epimers were the major compounds. Pasteurization produced a decrease in antioxidant capacity of fermented juice. However, TEAC (5.45 mM) and ORAC (6353 µM) values of orange beverage were similar to those of original orange juice. The novel orange beverage could be a valuable source of bioactive compounds with antioxidant capacity and exert potential beneficial effects.

Nicotinamide Riboside Is a Major NAD+ Precursor Vitamin in Cow Milk.

BACKGROUND: Nicotinamide riboside (NR) is a recently discovered NAD(+) precursor vitamin with a unique biosynthetic pathway. Although the presence of NR in cow milk has been known for more than a decade, the concentration of NR with respect to the other NAD(+) precursors was unknown. OBJECTIVE: We aimed to determine NAD(+) precursor vitamin concentration in raw samples of milk from individual cows and from commercially available cow milk. METHODS: LC tandem mass spectrometry and isotope dilution technologies were used to quantify NAD(+) precursor vitamin concentration and to measure NR stability in raw and commercial milk. Nuclear magnetic resonance (NMR) spectroscopy was used to test for NR binding to substances in milk. RESULTS: Cow milk typically contained ∼12 µmol NAD(+) precursor vitamins/L, of which 60% was present as nicotinamide and 40% was present as NR. Nicotinic acid and other NAD(+) metabolites were below the limits of detection. Milk from samples testing positive for Staphylococcus aureus contained lower concentrations of NR (Spearman ρ = -0.58, P = 0.014), and NR was degraded by S. aureus Conventional milk contained more NR than milk sold as organic. Nonetheless, NR was stable in organic milk and exhibited an NMR spectrum consistent with association with a protein fraction in skim milk. CONCLUSIONS: NR is a major NAD(+) precursor vitamin in cow milk. Control of S. aureus may be important to preserve the NAD(+) precursor vitamin concentration of milk.

A novel ß-carotene-associated carrot (Daucus carota L.) pectic polysaccharide.

OBJECTIVE: Carrot (Daucus carota L.) is a nutritional source with enriched ß-carotene (0.01%) and pectins (0.8%). Although studies have highlighted the association between ß-carotene and carrot pectic polysaccharide (CRPP), the precise binding of ß-carotene to CRPP and its biological implications were not yet clearly understood. The aim of the present study is to report for the first time a natural carbohydrate polymer-CRPP-with associated ß-carotene. METHODS: The presence of ß-carotene in CRPP and its binding is demonstrated by various studies including differential extraction followed by liquid chromatography (LC), liquid chromatography mass spectrometry (LC MS), and Fourier transform infrared spectroscopy (FTIR). Further, we show the binding via its release from CRPP using LC, fermentation studies and scanning electron microscopy (SEM) studies. The role of ß-carotene in induction of apoptosis and the galactic-3 inhibitory property was also studied. RESULTS: The presence of ß-carotene in CRPP was demonstrated, as well as an association between ß-carotene and CRPP, apoptotic and galectin-3 inhibitory properties of CRPP, and upregulation of connexin 43 by CRPP. CONCLUSION: The data highlights that with the combination of ß-carotene with CRPP, the prooxidant effect of ß-carotene may be reduced, an apoptotic effect may be enhanced, and the anticancer potential of CRPP thus may be strengthened via galectin-3 inhibition.