Christopher R Davis

The xanthophyll composition of biofortified maize (Zea mays Sp.) does not influence the bioefficacy of provitamin a carotenoids in Mongolian gerbils (Meriones unguiculatus).

Maize has been targeted for biofortification with provitamin A carotenoids through traditional breeding. Two studies were conducted in gerbils to evaluate factors that may affect provitamin A activity. Maize diets had equal theoretical concentrations of vitamin A (VA) assuming 100% bioefficacy. Study 1 ( n = 57) varied the ratio of beta-cryptoxanthin and beta-carotene but maintained the same theoretical VA. Study 2 ( n = 67) varied lutein and zeaxanthin. Other treatments were oil, VA, or beta-carotene doses. Serum and livers were analyzed for VA and carotenoids. In study 1, total liver VA did not differ among the maize groups. In study 2, total liver VA of the VA and maize groups were higher than controls ( P < 0.05). Conversion factors were 2.1-3.3 mug beta-carotene equivalents to 1 mug retinol. Twice the molar amount of beta-cryptoxanthin was as efficacious as beta-carotene and the proportion of beta-cryptoxanthin or xanthophylls did not appreciably change the VA value of biofortified maize.

β-Cryptoxanthin biofortified maize (Zea mays) increases β-cryptoxanthin concentration and enhances the color of chicken egg yolk

The laying hen has a natural ability to deposit carotenoids into its egg yolks, especially the xanthophyll carotenoid lutein that is used commercially as an egg colorant. Can this ability to deposit carotenoids be used to enrich egg yolk provitamin A value? After a 10-d carotenoid depletion period in hens (n = 24), the effects of a 20-d intervention with high-β-cryptoxanthin, high-β-carotene, or typical yellow maize on color and carotenoid profile were compared with the effects of a white maize diet (n = 6/treatment). Eggs were collected every other day and yolks were analyzed by using a portable colorimeter to define the color space and by using an HPLC to determine the carotenoid profile. The high-β-cryptoxanthin and yellow maize increased β-cryptoxanthin in the yolk (0.55 ± 0.08 to 4.20 ± 0.56 nmol/g and 0.55 ± 0.08 to 1.06 ± 0.12 nmol/g, respectively; P < 0.001). Provitamin A equivalents increased in eggs from hens fed high-β-cryptoxanthin maize (P < 0.001) but not the high-β-carotene maize. The color (L*, a*, and b*) assessment of the yolks showed an increase in the high-β-cryptoxanthin treatment for the red-green a* scale (P < 0.001) and a decrease for the light-dark L* scale (P < 0.001). No appreciable change was noted in the yellow-blue b* scale for the high-β-cryptoxanthin treatment; but significant changes were noted for the yellow (P = 0.002) and high-β-carotene maize (P = 0.005) treatments, which were most evident at the end of the washout period with white maize. β-Cryptoxanthin-biofortified maize is a potential vehicle to elevate provitamin A equivalents and to enhance the color of yolks. This could lead to a human health benefit if widely adopted.

Vitamin A isotope dilution predicts liver stores in line with long-term vitamin A intake above the current Recommended Dietary Allowance for young adult women

BACKGROUND The Estimated Average Requirement (EAR) and Recommended Dietary Allowance (RDA) for vitamin A are 1.7 and 2.4 μmol/d (500 and 700 μg retinol activity equivalents/d), respectively, for nonpregnant, nonlactating women aged >19 y. This intake is presumed to maintain a minimally acceptable liver concentration of 0.07 μmol (20 μg) retinol/g; however, liver reserves have not been evaluated with respect to vitamin A intake in women of any age group defined in the Dietary Reference Intakes. OBJECTIVE This cross-sectional study examined vitamin A intake and liver reserves estimated by stable-isotope dilution testing. DESIGN Forty nonpregnant, nonlactating women (mean ± SD age: 22.4 ± 2.3 y) completed a Harvard food-frequency questionnaire (FFQ) and 3-d diet record (3DDR) before undergoing vitamin A status assessment by using a [(13)C2]retinol stable-isotope dilution test. RESULTS Vitamin A intake was 70% higher than the RDA by both dietary-assessment methods (P < 0.001). The mean (±SD) liver concentration of vitamin A was 0.45 ± 0.31 μmol/g (129 ± 89 μg/g) and ranged from 0.09 (26 μg/g) to 1.79 μmol/g (513 μg/g). Liver and total-body vitamin A were highly correlated with intake measured by FFQ (P ≤ 0.009), but 3DDR was not (P ≥ 0.22). Prediction equations were developed for 3- and 7-d data. CONCLUSIONS In this well-nourished population, vitamin A consumption was considerably higher than recommended, and liver reserves were consistent with intake. Because of their sensitivity, stable-isotope techniques can help to describe the vitamin A status and better characterize the intake needs of all groups defined in the Dietary Reference Intakes. Registration was not required for this trial.

Carotenoid profiles in provitamin A-containing fruits and vegetables affect the bioefficacy in Mongolian gerbils

Fruits and vegetables are rich sources of provitamin A carotenoids. We evaluated the vitamin A (VA) bioefficacy of a whole foods supplement (WFS) and its constituent green vegetables (Study 1) and a variety of fruits with varying ratios of provitamin A carotenoids (Study 2) in VA-depleted Mongolian gerbils (n = 77/study). After feeding a VA-deficient diet for 4 and 6 weeks in Studies 1 and 2, respectively, customized diets, equalized for VA, were fed for 4 and 3 weeks, respectively. Both studies utilized negative and VA-positive control groups. In Study 1, liver VA was highest in the VA group (0.82 ± 0.16 μmol/liver, P < 0.05), followed by brussels sprouts (0.50 ± 0.15 μmol/liver), Betanat® (β-carotene from Blakeslea trispora) (0.50 ± 0.12 μmol/liver) and spinach (0.47 ± 0.09 μmol/liver) groups, which did not differ from baseline. The WFS (0.44 ± 0.06 μmol/liver) and kale (0.43 ± 0.14 μmol/liver) groups had lower liver VA than the baseline group (P < 0.05), but did not differ from the brussels sprouts, Betanat® and spinach groups. In Study 2, liver VA was highest in the orange (0.67 ± 0.18 μmol/liver), papaya (0.67 ± 0.15 μmol/liver) and VA (0.66 ± 0.14 μmol/liver) groups, followed by the mango (0.58 ± 0.09 μmol/liver) and tangerine (0.55 ± 0.15 μmol/liver) groups. These groups did not differ from baseline. The banana group (0.47 ± 0.15 μmol/liver) was unable to maintain baseline stores of VA and did not differ from the control (0.46 ± 0.13 μmol/liver). These fruits (except banana), vegetables and the WFS were able to prevent VA deficiency in Mongolian gerbils and could be an effective part of food-based interventions to support VA nutrition in developing countries and worldwide.

α-Retinol Is Distributed through Serum Retinol-Binding Protein-Independent Mechanisms in the Lactating Sow-Nursing Piglet Dyad

α-Retinol (αR) is a structural isomer of retinol [vitamin A (VA)] that does not bind to serum retinol-binding protein (RBP). In this study, α-retinyl acetate (αRA) was synthesized and given orally (35 μmol) to VA-deficient lactating sows (n = 11) to assess its potential to trace RBP-independent retinol transport and tissue uptake. The αRA dose primarily appeared in sow serum as 4 α-retinyl esters (αRE) with peak serum total αR concentrations (the sum of the alcohol and ester forms) detected at 2 h (70 ± 23 nmol/L, mean ± SEM) postdose. From 0 to 40 h postdose, the percentage of serum total αR in the alcohol form did not increase. Rapid αR uptake into sow milk was observed with peak concentrations (371 ± 83 nmol/L) at 7.5 h postdose, consistent with the uptake of αRE from chylomicra. A high percentage of the αRA dose (62 ± 15%, mean ± SD) was present in the livers of sows (n = 6) killed 22-28 d postdose. Approximately 15-26% of the sow αRA dose was transferred to the livers of the nursing piglets (n = 17) after 3 d. In piglets and sows, a similar percentage of hepatic total αR was detected in the ester form as that of hepatic total retinol. Taken together, these data suggest that an oral dose of αRA effectively traces the uptake, esterification, chylomicron transport, and hepatic storage of retinol and may be useful for deciphering the role of RBP-independent delivery of retinol to other tissues.

Relative vitamin A values of 9- cis - and 13- cis -β-carotene do not differ when fed at physiological levels during vitamin A depletion in Mongolian gerbils ( Meriones unguiculatus )

Provitamin A biofortification of staple crops may decrease the prevalence of vitamin A (VA) deficiency if widely adopted in target countries. To assess the impact of processing methods on the VA value of plant foods, the unique bioefficacies of cis-βC isomers (formed during cooking) compared with all-trans (at) β-carotene (βC) must be determined. The bioefficacies of 9-cis (9c)- and 13-cis (13c)-βC isomers were compared with those of the at-βC isomer and VA positive (VA+) and negative (VA - ) controls in VA-depleted Mongolian gerbils (Meriones unguiculatus) in two experimental studies (study 1, n 56; study 2, n 57). A 3- or 4-week depletion period was followed by a 3- or 4-week treatment period in which the groups received oral doses of the 9c-, 13c- or at-βC isomers in cottonseed oil (study 1, 15 nmol/d; study 2, 30 nmol/d). In study 1, the βC isomers did not maintain baseline liver VA stores in all groups (0.69 (SD 0.20) μmol/liver) except in the VA+group (0.56 (SD 0.10) μmol/liver) (P= 0.0026). The βC groups were similar to the VA+group, but the 9c- and 13c-βC groups did not differ from the VA - group (0.39 (SD 0.09) μmol/liver). In study 2, the βC isomers maintained baseline liver VA stores in all the βC groups (0.35 (SD 0.13) μmol/liver), and in the VA+group, the VA supplement (0.54 (SD 0.19) μmol/liver) exceeded the baseline VA status (0.38 (SD 0.15) μmol/liver) (P< 0.0001); however, the 9c-βC group did not differ from the VA - group (0.20 (SD 0.07) μmol/liver). In vivo isomerisation of βC was confirmed in both experimental studies. Lower VA bioconversion factor values were obtained for the cis-βC isomers in study 2 when compared with study 1, but higher values were obtained for the at-βC isomer. Dose and VA status clearly affect bioconversion factors. In conclusion, the cis-βC isomers yielded similar liver VA stores to the at-βC isomer in Mongolian gerbils, and liver VA stores of the 9c- and 13c-βC groups did not differ when the doses were provided at physiological levels over time in two studies.

Provitamin A-biofortified maize consumption increases serum xanthophylls and 13C-natural abundance of retinol in Zambian children

Plants that undergo C4 photosynthesis, such as maize, are enriched in the stable isotope of carbon (13C) compared with other dietary plants and foods. Consumption of maize that has been biofortified to contain elevated levels of provitamin A carotenoids (orange maize) increased the abundance of 13C in serum retinol of Mongolian gerbils. We evaluated this method in humans to determine if it has potential for further use in intervention effectiveness studies. A random subset of samples from a two-month randomized controlled feeding trial of rural three- to five-year old Zambian children were used to determine the impact of orange maize intake on serum carotenoid concentrations (n = 88) and 13C-natural abundance in serum retinol (n = 77). Concentrations of β-cryptoxanthin (a xanthophyll provitamin A carotenoid) and the dihydroxy xanthophylls lutein and zeaxanthin, which do not have vitamin A activity, were elevated in children consuming orange maize compared with those consuming a white maize control (P < 0.001), while β-carotene was not different (P > 0.3). Furthermore, 13C natural abundance was higher after two months’ intervention in the orange maize group compared with the white maize group (P = 0.049). Predictions made from equations developed in the aforementioned gerbil study estimated that maize provided 11% (2–21%, 95% confidence interval) of the recent dietary vitamin A to these children. These results demonstrate that orange maize is efficacious at providing retinol to the vitamin A pool in children through provitamin A carotenoids, as monitored by the change in 13C enrichment, which was not reflected in serum β-carotene concentrations. Further effectiveness studies in countries who have adopted orange maize should consider determining differences in retinol 13C-enrichment among target groups in addition to profiling serum xanthophyll carotenoids with specific emphasis on zeaxanthin. Impact statement Maize biofortified with provitamin A carotenoids (orange) has been released in some African markets. Responsive and sensitive methods to evaluate dissemination effectiveness are needed. This study investigated methods to evaluate effectiveness of orange maize consumption using serum from Zambian children fed orange maize for two months. Many varieties of orange maize contain higher amounts of the xanthophyll carotenoids in addition to β-carotene compared with typical varieties. This study uniquely showed higher concentrations of the maize xanthophylls lutein, zeaxanthin, and β-cryptoxanthin in children who consumed orange maize compared with white. Furthermore, maize is a C4 plant and is therefore naturally enriched with 13C. Higher 13C was detected in the serum retinol of the orange maize consumers with no change in serum β-carotene concentration suggesting preferential bioconversion to retinol. The combined analyses of serum zeaxanthin specifically and 13C-natural abundance of retinol could prove useful in effectiveness studies between orange maize adopters and non-adopters.

Retention of Carotenoids in Biofortified Maize Flour and β-Cryptoxanthin-Enhanced Eggs after Household Cooking

Biofortification of crops to enhance provitamin A carotenoids is a strategy to increase the intake where vitamin A deficiency presents a widespread problem. Heat, light, and oxygen cause isomerization and oxidation of carotenoids, reducing provitamin A activity. Understanding provitamin A retention is important for assessing efficacy of biofortified foods. Retention of carotenoids in high-xanthophyll and high-β-carotene maize was assessed after a long-term storage at three temperatures. Carotenoid retention in high-β-cryptoxanthin maize was determined in muffins, non-nixtamalized tortillas, porridge, and fried puffs made from whole-grain and sifted flour. Retention in eggs from hens fed high-β-cryptoxanthin maize was assessed after frying, scrambling, boiling, and microwaving. Loss during storage in maize was accelerated with increasing temperature and affected by genotype. Boiling whole-grain maize into porridge resulted in the highest retention of all cooking and sifting methods (112%). Deep-fried maize and scrambled eggs had the lowest carotenoid retention rates of 67–78 and 84–86%, respectively.

13C Natural Abundance of Serum Retinol Is a Novel Biomarker for Evaluating Provitamin A Carotenoid-Biofortified Maize Consumption in Male Mongolian Gerbils

Background: Crops such as maize, sorghum, and millet are being biofortified with provitamin A carotenoids to ensure adequate vitamin A (VA) intakes. VA assessment can be challenging because serum retinol concentrations are homeostatically controlled and more sensitive techniques are resource-intensive. Objectives: We investigated changes in serum retinol relative differences of isotope amount ratios of 13C/12C (δ13C) caused by natural 13C fractionation in C3 compared with C4 plants as a biomarker to detect provitamin A efficacy from biofortified (orange) maize and high-carotene carrots. Methods: The design was a 2 × 2 × 2 maize (orange compared with white) by carrot (orange compared with white) by a VA fortificant (VA+ compared with VA−) in weanling male Mongolian gerbils (n = 55), which included a 14-d VA depletion period and a 62-d treatment period (1 baseline and 8 treatment groups; n = 5−7/group). Liver VA and serum retinol were quantified, purified by HPLC, and analyzed by GC combustion isotope ratio mass spectrometry for 13C. Results: Treatments affected liver VA concentrations (0.048 ± 0.039 to 0.79 ± 0.24 μmol/g; P < 0.0001) but not overall serum retinol concentrations (1.38 ± 0.22 μmol/L). Serum retinol and liver VA δ13C were significantly correlated (R2 = 0.92; P < 0.0001). Serum retinol δ13C differentiated control groups that consumed white maize and white carrots (−27.1 ± 1.2 δ13C‰) from treated groups that consumed orange maize and white carrots (−21.6 ± 1.4 δ13C‰ P < 0.0001) and white maize and orange carrots (−30.6 ± 0.7 δ13C‰ P < 0.0001). A prediction model demonstrated the relative contribution of orange maize to total dietary VA for groups that consumed VA from mixed sources. Conclusions: Provitamin A efficacy and quantitative estimation of the relative contribution to dietary VA were demonstrated with the use of serum retinol δ13C. This method could be used for maize efficacy or effectiveness studies and with other C4 crops biofortified with provitamin A carotenoids (e.g., millet, sorghum). Advantages include no extrinsic tracer dose, 1 blood sample, and higher sensitivity than serum retinol concentrations alone.

Interspecies comparison of stellate cell-containing macula flavae and vitamin A storage in vocal fold mucosa

The macula flavae (MF), populated by vitamin A‐storing stellate cells (SCs), are believed to play a fundamental role in development, maintenance and repair of the vocal fold (VF) mucosa; however, to date, they have mostly been examined in observational human cadaver studies. Here, we conducted an interspecies comparison of MF and SC phenotype, as well as vitamin A quantification and localization, in human, pig, dog, rabbit and rat VF mucosae. MF containing vitamin A‐positive SCs were only identified in human and rat specimens. Pig, dog and rabbit VF mucosae contained no discernable MF, but rather exhibited preferential vitamin A localization to mucous (pig), serous (dog) or mixed (rabbit) glands. This glandular vitamin A storage corresponded to exceedingly high concentrations of retinol in pig and dog mucosae, and retinyl ester in dog mucosa. These findings have significant implications for the presumed role of the MF and SCs in VF biology, the nature of vitamin A storage within the VF mucosa, and the selection of an appropriate animal model for future experimental studies.